CN113049477B

CN113049477B - Apparatus and method for cell analysis

Info

Publication number: CN113049477B
Application number: CN202110216932.XA
Authority: CN
Inventors: 施文典; 孟兆恺; 丁宇喆
Original assignee: Core Easy Diagnosis Co ltd
Current assignee: Core Easy Diagnosis Co ltd
Priority date: 2017-06-14
Filing date: 2018-06-12
Publication date: 2024-05-21
Anticipated expiration: 2038-06-12
Also published as: CN113049476B; CN113049476A; US20220082486A1; US20200158615A1; US11215545B2; EP3638987A1; CN110753830A; WO2018231835A1; CN110753830B; EP3638987A4; US20230417643A1; US11768143B2; CN113049477A

Abstract

The present disclosure relates to devices, device systems, and methods for analyzing cells (e.g., blood cells) and/or particles in a sample. The present disclosure provides various devices and device systems, which may include: a light source, a condenser lens, and one, two or more detectors. These devices and device systems may also include a flow chamber or a cartridge device including a flow chamber. The present disclosure provides various methods, which may include the steps of: using a light source to emit illumination light; illuminating the sample stream with the illumination light; and one, two or more detectors are used to detect the collected scattered light and fluorescence. The methods may include forming a sample stream using a flow cell. The methods may further comprise the steps of: receiving a sample into a cartridge device having a flow chamber; mixing the sample with the reagent using the cartridge device to form a measurement sample; and using the flow cell to form a sample stream of the measurement sample.

Description

Apparatus and method for cell analysis

The application is a divisional application of patent application with application date 2018, month 06 and 12, application number 2018800397417 and the application name of a device and a method for cell analysis.

Priority statement

The present application claims priority from U.S. provisional patent application No. 62/519,467 entitled "apparatus and method for cell analysis (DEVICES AND Methods for CELL ANALYSIS)" filed on date 6 and 14 in 2017, which is incorporated herein by reference in its entirety.

Technical Field

The present disclosure relates to drugs, cell counts and medical devices.

Background

All publications cited herein are incorporated by reference in their entirety as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be used to understand the present disclosure. It is not an admission that any of the information provided herein is prior art or that any publication relevant to this disclosure or specifically or implicitly referenced is prior art.

Flow cytometry (flow cytometry) is a powerful method for detecting cells in a sample and analyzing their characteristics with high throughput. By forming a sample stream within the flow chamber and illuminating the sample stream with light from a light source, signals such as scattered light with a forward angle, scattered light with a side angle, and fluorescence can be detected from individual cells and used to analyze characteristics of the cells such as cell size, cell granularity, cell nucleic acid, cell membrane integrity, cell antigen expression, and the like.

In clinical applications, flow cytometry has been widely used to detect and analyze cells in human or animal blood, such as counting the number of blood cells, classifying blood cells into different types (e.g., white blood cells, red blood cells, and platelets), and analyzing antigen expression of cells (e.g., CD4 ⁺ antigen, CD8 ⁺ antigen, etc.). For example, in blood analysis, flow cytometry has been used to measure the total number of leukocytes, the total number of erythrocytes, and the total number of platelets in each sample volume, and to further divide the leukocytes into different subtypes (e.g., lymphocytes, monocytes, neutrophils, eosinophils, and basophils) and determine their respective percentages. For another example, flow cytometry is used to count the number of CD4 ⁺ lymphocytes and CD8 ⁺ lymphocytes in blood in AIDS diagnosis.

Conventional analyzers for flow cytometric analysis typically have a fixed flow chamber to form a sample stream. The alignment between the flow cell and the optical detection component is fixed and has no deviation. In contrast, in analyzers with replaceable or disposable flow cells, the alignment between the flow cell and the optical detection component may have significant deviations whenever the flow cell is replaced. This misalignment can be a significant problem for analyzers in which the flow cell is handled and replaced after each sample measurement.

In addition, flow cells in conventional analyzers typically have at least two optically transparent surfaces for signal detection, one of which is used to measure scattered light from the sample flow having a forward angle (e.g., a scatter angle less than about 20 degrees), and the other of which is used to measure scattered light from the sample flow having a side angle (e.g., a scatter angle greater than about 70 degrees), and fluorescence, or both. However, in some analyzers, the flow cell (e.g., some low cost flow cells made by plastic injection molding processes) may have only one optically transparent surface for signal detection. Low cost replaceable or disposable flow chambers are necessary for many applications such as point-of-care diagnostics. A flow cell having only one optically transparent surface for signal detection may limit the choice of detectable signals.

In addition, replaceable or disposable flow cells are typically built into cartridge devices, and the surfaces of the cartridge devices or any other surface in the optical path may reflect light back to the light source and introduce unwanted noise. Moreover, the intensity of the optical signal detected from targets (e.g., particles and cells) within the replaceable or disposable flow chamber can be weak, thus increasing the collection efficiency of the optical signal can be challenging.

To address these challenges, the present disclosure provides various devices and methods for analyzing particles and cells.

Disclosure of Invention

The following embodiments and aspects thereof will be described and illustrated in conjunction with devices, systems and methods, which are meant to be exemplary and illustrative, not limiting in scope.

To address those challenges discussed above, the present disclosure provides various devices and methods for analyzing particles and cells. In various embodiments, the present disclosure provides various devices and methods for analyzing cells in a blood sample. In various embodiments, these devices and methods may also be used to analyze other particles (e.g., beads, nanoparticles, protein molecules, nucleic acid molecules, etc.) in a sample. In various embodiments, these devices and methods are applicable to replaceable or disposable flow cells. In various embodiments, these devices and methods are applicable to flow cells having only one optically transparent indicator for signal detection and measurement. In various embodiments, these devices and methods are also compatible with other flow cells, such as fixed flow cells and flow cells having more than one optically transparent surface for signal detection and measurement.

Various embodiments of the present disclosure provide an apparatus or apparatus system comprising: a light source configured to emit illumination light for illuminating a sample stream, wherein the sample stream comprises particles and/or cells; a condensing lens configured to collect both scattered light having a forward angle and fluorescence from particles and/or cells in the sample stream; and one, two or more detectors configured to detect the signal of scattered light having a forward angle and the signal of fluorescence. The device or device system as disclosed herein may be used to analyze particles and/or cells in a sample.

Various embodiments of the present disclosure provide an apparatus or apparatus system comprising: a flow chamber configured to form a sample stream of a measurement sample, the measurement sample comprising particles and/or cells; a light source configured to emit illumination light for illuminating the sample stream; a condensing lens configured to collect both scattered light having a forward angle and fluorescence from particles and/or cells in the sample stream; and one, two or more detectors configured to detect the signal of scattered light having a forward angle and the signal of fluorescence. The device or device system as disclosed herein may be used to analyze particles or/and cells in a sample.

Various embodiments of the present disclosure provide a method of analyzing particles and/or cells in a sample stream, comprising: using a light source to emit illumination light; illuminating the sample stream with the illumination light; using a condensing lens to collect scattered light having a forward angle from particles and/or cells in the sample stream as well as fluorescence; and using one, two or more detectors to detect the signal of scattered light with a forward angle and the signal of fluorescence.

Various embodiments of the present disclosure provide a method of analytically measuring particles and/or cells in a sample, comprising: forming a sample stream of the measurement sample using a flow cell; using a light source to emit illumination light; illuminating the sample stream with the illumination light; using a condensing lens to collect scattered light having a forward angle from particles and/or cells in the sample stream as well as fluorescence; and using one, two or more detectors to detect the signal of scattered light with a forward angle and the signal of fluorescence.

Various embodiments of the present disclosure provide an apparatus or apparatus system comprising: a light source configured to emit illumination light for illuminating the sample stream, wherein the sample stream comprises particles and/or cells; a condensing lens configured to collect both scattered light with a side angle and fluorescence from particles and/or cells in the sample stream; and one, two or more detectors configured to detect the signal of the scattered light having a lateral angle and the signal of the fluorescence. The device or device system as disclosed herein may be used to analyze particles and/or cells in a sample.

Various embodiments of the present disclosure provide an apparatus or apparatus system comprising: a flow chamber configured to form a sample stream of a measurement sample, the measurement sample comprising particles and/or cells; a light source configured to emit illumination light for illuminating the sample stream; a condensing lens configured to collect both scattered light with a side angle and fluorescence from particles and/or cells in the sample stream; and one, two or more detectors configured to detect the signal of the scattered light having a lateral angle and the signal of the fluorescence. The device or device system as disclosed herein may be used to analyze particles and/or cells in a sample.

Various embodiments of the present disclosure provide a method of analyzing particles and/or cells in a sample stream, comprising: using a light source to emit illumination light; illuminating the sample stream with the illumination light; using a condensing lens to collect scattered light from particles and/or cells in the sample stream having a lateral angle and fluorescence; and using one, two or more detectors to detect the signal of scattered light with a lateral angle and the signal of fluorescence.

Various embodiments of the present disclosure provide a method of analytically measuring particles and/or cells in a sample, comprising: forming a sample stream of the measurement sample using the flow chamber; using a light source to emit illumination light; illuminating the sample stream with the illumination light; using a condensing lens to collect scattered light from particles and/or cells in the sample stream having a lateral angle and fluorescence; and using one, two or more detectors to detect the signal of scattered light with a lateral angle and the signal of fluorescence.

Various embodiments of the present disclosure provide methods for analyzing particles and/or cells in a sample, comprising: receiving the sample into a cartridge device having a flow chamber; mixing the sample with a reagent using the cartridge device to form a measurement sample; forming a sample stream of the measurement sample using the flow chamber; using a light source to emit illumination light; illuminating the sample stream with the illumination light; using a condensing lens to collect both scattered light from particles and/or cells in the sample stream as well as fluorescence; and one, two or more detectors are used to detect the signal of the scattered light and the signal of the fluorescence. In some embodiments, the scattered light comprises forward scattered light, i.e., scattered light having a forward angle (e.g., a scatter angle of less than about 25 degrees). In other embodiments, the scattered light comprises side-scattered light, i.e., scattered light having a side angle (e.g., a scattering angle greater than about 25 degrees).

Drawings

Exemplary embodiments are illustrated in referenced figures. The embodiments and figures disclosed herein are to be regarded as illustrative rather than restrictive.

Fig. 1A shows one non-limiting example of a device or device system as disclosed herein, including a sample supply unit, a detection unit, and a signal analysis unit, according to various embodiments of the present disclosure.

Fig. 1B illustrates one non-limiting example of a detection unit, including a light source, a focusing module, a flow cell, a condensing lens, a receiving module, and a detector, according to various embodiments of the present disclosure.

Fig. 2A and 2B illustrate one non-limiting example of a detection unit in which a flow cell is used to form a sample flow of a measurement sample, according to various embodiments of the present disclosure.

Fig. 3A illustrates that when blocking illumination light, the beam blocker also blocks scattered light having a scatter angle less than θ ₁, according to various embodiments of the present disclosure.

Fig. 3B illustrates that the beam blocker may be a light blocking strip comprising an opaque material in accordance with various embodiments of the present disclosure.

Fig. 4A illustrates that a beam blocker may be positioned between a flow chamber and a condenser lens according to various embodiments of the present disclosure.

Fig. 4B illustrates that a beam blocker may be positioned behind a condenser lens according to various embodiments of the present disclosure.

Fig. 5A illustrates a spherical lens having at least one curved surface with a spherical shape, according to various embodiments of the present disclosure.

Fig. 5B illustrates an aspherical lens having at least one curved surface with an aspherical shape defined by an equation, according to various embodiments of the present disclosure.

Fig. 6A and 6B illustrate that a flow cell may be positioned at or near the focal point of one lens (fig. 6A) and away from the focal point of the other lens (fig. 6B), in accordance with various embodiments of the present disclosure.

Fig. 6C and 6D illustrate that the diameter of the elliptical spot along the sample flow direction (D ₁) (i.e., the diameter along the y-axis) is narrow and the diameter of the elliptical spot perpendicular to the sample flow direction (D ₂) (i.e., the diameter along the x-axis) is wide, according to various embodiments of the present disclosure.

Fig. 7A illustrates one non-limiting example in which a circular spot is used to illuminate a sample stream, according to various embodiments of the present disclosure.

Fig. 7B illustrates that when the spot has an alignment deviation Δx from the sample stream, the spot is no longer able to illuminate the sample stream, in accordance with various embodiments of the present disclosure.

Fig. 7C illustrates one non-limiting example in which an elliptical spot is used to illuminate the sample stream, according to various embodiments of the present disclosure.

Fig. 7D illustrates that the spot is still capable of illuminating the sample stream with the same alignment deviation Δx, according to various embodiments of the present disclosure.

Fig. 8 illustrates an enlarged view of the detection unit of fig. 2B, according to various embodiments of the present disclosure.

Fig. 9A and 9B illustrate another non-limiting example of obtaining an elliptical spot according to various embodiments of the present disclosure, wherein the focusing module 903 includes a beam focusing lens 909 and a cylindrical lens 911.

Fig. 10A illustrates a flow cell with a sheath flow that can be used, wherein a sample flow is surrounded by the sheath flow within the flow cell, in accordance with various embodiments of the present disclosure.

Fig. 10B illustrates a flow cell without sheath flow that can be used, where no sheath flow surrounds the sample flow within the flow cell, in accordance with various embodiments of the present disclosure.

FIG. 11 illustrates one non-limiting example of a light source including a light emitting component, an optical fiber, and a beam focusing lens, according to various embodiments of the present disclosure.

FIG. 12 shows one non-limiting example of a scatter plot of fluorescence and scattered light, where each point represents one white blood cell being detected in a sample stream, according to various embodiments of the present disclosure.

Fig. 13 shows one non-limiting example of a histogram in which the intensity of fluorescence is plotted as the x-axis and the number of detected cells having fluorescence of the corresponding intensity is plotted as the y-axis, according to various embodiments of the present disclosure.

FIG. 14 shows one non-limiting example of a scatter plot of fluorescence and scattered light, where each point represents one red blood cell or platelet being detected in a sample stream, according to various embodiments of the present disclosure.

Fig. 15 shows one non-limiting example of a histogram in which the intensity of scattered light is plotted as the x-axis and the number of detected cells with corresponding intensities of scattered light is plotted as the y-axis, according to various embodiments of the present disclosure.

Fig. 16A and 16B illustrate another non-limiting example of a detection unit according to various embodiments of the present disclosure.

Fig. 16C illustrates an enlarged view of a light source, a focusing module, and a flow chamber, according to various embodiments of the present disclosure. In this non-limiting example, the optical axis of the spherical lens 1605 and the optical axis of the cylindrical lens 1606 are coaxial, and they are also coaxial with the central axis 1616 of the light emitted from the light source.

Fig. 16D illustrates an enlarged view of a light source, a focusing module, and a flow chamber, according to various embodiments of the present disclosure. In this non-limiting example, the optical axis of the spherical lens 1605 and the optical axis of the cylindrical lens 1606 are coaxial, but they are not coaxial with the central axis 1616 of the light emitted from the light source.

Fig. 16E illustrates one non-limiting example of a detection unit in which two lenses 1605 and 1606 are coaxial with each other, but they are not coaxial with a central axis 1616 of light emitted from light source 1602, according to various embodiments of the present disclosure.

Fig. 16F illustrates one non-limiting example of a detection unit in which the lens 1606 is coaxial with the central axis 1616 of the emitted light, but the lens 1605 is not coaxial with the central axis 1616 of the emitted light, according to various embodiments of the present disclosure.

Fig. 16G illustrates an enlarged view of a light source, a focusing module, and a flow chamber, according to various embodiments of the present disclosure. In this non-limiting example, the flow cell 1601 is tilted in such a way that the surface 1621 of the flow cell is not perpendicular to the illuminating light 1617. For example, the angle θ ₃ between the surface 1621 and the central axis 1616 of the illumination light 1617 is not equal to 90 degrees.

Fig. 17 illustrates an example embodiment of a doublet that may be used with any of the embodiments disclosed herein.

Fig. 18A and 18B illustrate one non-limiting example of a detection unit in which a doublet is used to improve collection efficiency, according to various embodiments of the present disclosure.

Detailed Description

All references cited herein are incorporated by reference in their entirety as if fully set forth herein. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Tabelling, ministry of micro-fluid mechanics (reprint), (Introduction to Microfluidics), oxford university press (2010); hguyen et al, basic principles and applications of microfluidics (2 nd edition) (Fundamentals and Applications of Microfluidics nd ed.), artech House Incorporated (2006); berg et al, microfluidics for medical applications (Microfluidics for Medical Applications), royal society of chemistry (2014); gomez et al, biological application of microfluidics (1 st edition) (Biological Applications of Microfluidics st ed.), wiley-Interscience (2008); and Colin et al, microfluidics (1 st ed.), wiley-ISTE (2010) provide one of ordinary skill in the art with a general guidance on many of the terms used in the present application.

Those skilled in the art will recognize that many methods and materials similar or equivalent to those described herein can be used in the practice of the present disclosure. Other features and advantages of the present disclosure will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various features of embodiments of the present disclosure. Indeed, the present disclosure is in no way limited to the described methods and materials. For convenience, certain terms used herein in the specification, examples, and appended claims are collected here.

Unless otherwise indicated or implied by the context, the following terms and phrases include the meanings provided below. Unless explicitly stated otherwise or apparent from the context, the following terms and phrases do not exclude the meaning that the term or phrase has in the art to which it pertains. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It is to be understood that this disclosure is not limited to the particular methodology, protocols, reagents, etc. described herein, as such may vary. Definitions and terms are used herein to aid in describing particular embodiments and are not intended to limit the claims.

As used herein, the terms "comprise" or "comprising" are used to refer to compositions, methods, and their respective components, which may be used in the examples, but include unspecified elements, whether useful or not. It will be appreciated by those skilled in the art that, in general, terms used herein are generally intended to be interpreted as "open" terms (e.g., the term "comprising" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "including" should be interpreted as "including but not limited to," etc.).

The use of the terms "a" and "an" and "the" and similar referents in the context of describing particular embodiments of the application (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein with respect to certain embodiments, is intended merely to better illuminate the application and does not pose a limitation on the scope of the application claimed. The abbreviation "e.g. (e.g.)" is derived from latin language exempli gratia and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g. (e.g.)" is synonymous with the term "e.g. (for example)". No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the application.

Various embodiments of the present disclosure provide an apparatus or apparatus system comprising: a light source configured to emit illumination light for illuminating a sample stream, wherein the sample stream comprises particles and/or cells; a condensing lens configured to collect both scattered light having a forward angle and fluorescence from particles and/or cells in the sample stream; and one, two or more detectors configured to detect a signal of scattered light and a signal of fluorescence having a forward angle. The device or device system as disclosed herein may be used to analyze particles and/or cells in a sample.

Various embodiments of the present disclosure provide an apparatus or apparatus system comprising: a flow chamber configured to form a sample flow of a measurement sample, the measurement sample comprising particles and/or cells; a light source configured to emit illumination light for illuminating the sample stream; a condensing lens configured to collect both scattered light having a forward angle and fluorescence from particles and/or cells in the sample stream; and one, two or more detectors configured to detect a signal of scattered light and a signal of fluorescence having a forward angle. The device or device system as disclosed herein may be used to analyze particles and/or cells in a sample.

Various embodiments of the present disclosure provide an apparatus or apparatus system comprising: a light source configured to emit illumination light for illuminating a sample stream, wherein the sample stream comprises particles and/or cells; a condensing lens configured to collect both scattered light with a lateral angle from particles and/or cells in the sample stream and fluorescence; and one, two or more detectors configured to detect a signal of scattered light and a signal of fluorescence having a lateral angle. The device or device system as disclosed herein may be used to analyze particles and/or cells in a sample.

Various embodiments of the present disclosure provide an apparatus or apparatus system comprising: a flow chamber configured to form a sample flow of a measurement sample, the measurement sample comprising particles and/or cells; a light source configured to emit illumination light for illuminating the sample stream; a condensing lens configured to collect both scattered light with a lateral angle from particles and/or cells in the sample stream and fluorescence; and one, two or more detectors configured to detect a signal of scattered light and a signal of fluorescence having a lateral angle. The device or device system as disclosed herein may be used to analyze particles and/or cells in a sample.

In various embodiments, the apparatus or apparatus system as described herein further comprises a focusing module configured to focus the illumination light to form an elliptical spot on the sample stream. In various embodiments, an apparatus or apparatus system as described herein further comprises a receiving module configured to split the scattered light and the fluorescent light collected by the condenser lens into two separate light paths towards two separate detectors, respectively. In various embodiments, an apparatus or apparatus system as described herein further comprises a doublet lens configured to focus the concentrated fluorescence light. In various embodiments, the device or device system as described herein further comprises a signal analysis unit configured to analyze the signal of scattered light and the signal of fluorescence to analyze the particles and/or cells.

Various embodiments of the present disclosure provide methods of analyzing particles and/or cells in a sample stream, comprising: using a light source to emit illumination light; illuminating the sample stream with the illumination light; using a condensing lens to collect both scattered light with a forward angle from particles and/or cells in the sample stream as well as fluorescence; and one, two or more detectors are used to detect the signal of scattered light and the signal of fluorescence with a forward angle.

Various embodiments of the present disclosure provide a method of analyzing particles and/or cells in a sample stream, comprising: using the flow chamber to form a sample stream of the measurement sample; using a light source to emit illumination light; illuminating the sample stream with illumination light; using a condensing lens to collect scattered light with a forward angle from particles and/or cells in the sample stream as well as fluorescence; and one, two or more detectors are used to detect the signal of scattered light and the signal of fluorescence with a forward angle. In some embodiments, two separate detectors are used to detect the signal of scattered light and the signal of fluorescence with a forward angle. In various embodiments, the method further comprises focusing the illumination light using a focusing module to form an elliptical spot on the sample stream. In various embodiments, the method further includes using a receiving module to split the scattered light with forward angle and the fluorescence collected by the condenser lens into two separate light paths towards two separate detectors, respectively. In various embodiments, the method further comprises focusing the collected fluorescence using a doublet lens. In various embodiments, the method further comprises analyzing the signal of scattered light and the signal of fluorescence with a forward angle using a signal analysis unit to analyze the particles and/or cells.

Various embodiments of the present disclosure provide methods of analyzing particles and/or cells in a sample stream, comprising: using a light source to emit illumination light; illuminating the sample stream with the illumination light; using a condensing lens to collect both scattered light with a lateral angle from particles and/or cells in the sample stream as well as fluorescence; and one, two or more detectors are used to detect the signal of scattered light and the signal of fluorescence with a lateral angle.

Various embodiments of the present disclosure provide a method of analyzing particles and/or cells in a sample stream, comprising: using the flow chamber to form a sample stream of the measurement sample; using a light source to emit illumination light; illuminating the sample stream with illumination light; using a condensing lens to collect scattered light with a lateral angle from particles and/or cells in the sample stream as well as fluorescence; and one, two or more detectors are used to detect the signal of scattered light and the signal of fluorescence with a lateral angle. In some embodiments, two separate detectors are used to detect the signal of scattered light and the signal of fluorescence with a lateral angle. In various embodiments, the method further comprises focusing the illumination light using a focusing module to form an elliptical spot on the sample stream. In various embodiments, the method further includes using the receiving module to split the scattered light with the side angles and the fluorescence collected by the condensing lens into two separate light paths towards two separate detectors, respectively. In various embodiments, the method further comprises focusing the collected fluorescence using a doublet lens. In various embodiments, the method further comprises analyzing the signal of scattered light and the signal of fluorescence with a lateral angle using a signal analysis unit to analyze the particles and/or cells.

Various embodiments of the present disclosure provide a method for analyzing particles and/or cells in a sample, comprising: receiving a sample into a cartridge device having a flow chamber; mixing the sample with the reagent using a cartridge device to form a measurement sample; using the flow chamber to form a sample stream of the measurement sample; using a light source to emit illumination light; illuminating the sample stream with illumination light; using a condensing lens to collect both scattered light from particles and/or cells in the sample stream as well as fluorescence; and one, two or more detectors are used to detect the signal of scattered light and the signal of fluorescence. In some embodiments, the scattered light comprises forward scattered light, i.e., scattered light having a forward angle (e.g., a scattering angle of less than about 25 degrees). In other embodiments, the scattered light comprises side-scattered light, i.e., scattered light having a side angle (e.g., a scattering angle greater than about 25 degrees). In various embodiments, two separate detectors are used to detect the signal of scattered light and the signal of fluorescence. In various embodiments, the method further comprises focusing the illumination light using a focusing module to form an elliptical spot on the sample stream. In various embodiments, the method further includes using a receiving module to split the scattered light and the fluorescent light collected by the condenser lens into two separate light paths toward two separate detectors, respectively. In various embodiments, the method further comprises focusing the collected fluorescence using a doublet lens. In various embodiments, the method further comprises analyzing the signal of scattered light and the signal of fluorescence using a signal analysis unit to analyze the particles and/or cells.

In various embodiments, the light source comprises a laser diode, a Light Emitting Diode (LED), a laser module, or a halogen lamp, or a combination of a laser diode, a Light Emitting Diode (LED), a laser module, and a halogen lamp. In some embodiments, the light source includes a laser diode and an optical fiber.

In various embodiments, the condenser lens comprises a spherical lens, an aspherical lens, or a doublet, or a combination of a spherical lens, an aspherical lens, and a doublet. In some embodiments, the condenser lens is a spherical lens. In some embodiments, the condenser lens is an aspherical lens. In some embodiments, the condensing lens is a doublet.

In various embodiments, an apparatus or apparatus system as disclosed herein includes two independent detectors: one detector is configured to detect a signal of scattered light having a forward angle and the other detector is configured to detect a signal of fluorescence. In various embodiments, an apparatus or apparatus system as disclosed herein includes two independent detectors: one detector is configured to detect a signal of scattered light having a lateral angle and the other detector is configured to detect a signal of fluorescence.

In various embodiments, an apparatus or apparatus system as disclosed herein includes a first detector configured to detect a signal of scattered light having a forward angle, and a second detector configured to detect a signal of fluorescence. In various embodiments, an apparatus or apparatus system as disclosed herein includes a first detector configured to detect a signal of scattered light having a lateral angle, and a second detector configured to detect a signal of fluorescence. The terms "first" and "second" are used to distinguish between different detectors according to the present disclosure, and they do not indicate any sequential relationship.

In various embodiments, the detector for fluorescence comprises a photodiode, an avalanche photodiode, or a silicon photomultiplier, or a combination of a photodiode, an avalanche photodiode, and a silicon photomultiplier.

In various embodiments, the concentrated scattered light comprises or consists of forward scattered light having a scatter angle of less than about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 degrees. In various embodiments, the forward scattered light that is detected comprises or consists of scattered light having a scatter angle of less than about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 degrees.

In various embodiments, the concentrated scattered light comprises or consists of side-scattered light having a scatter angle of greater than about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 degrees. In various embodiments, the scattered light that is detected comprises or consists of side-scattered light having a scatter angle of greater than about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 degrees.

In various embodiments, an apparatus or apparatus system as described herein further comprises a focusing module.

In some embodiments, the focusing module is configured to focus the illumination light to form an elliptical spot on the sample stream. In some embodiments, the focusing module includes a lens that is either non-coaxial or non-perpendicular to the central axis of the illuminating light.

In various embodiments, the elliptical spot has a width that is greater than the width of the flow cell. In various embodiments, the elliptical spot covers more than the entire width of the flow cell. In various embodiments, the long axis (d ₂) of the elliptical spot is perpendicular to the direction of the sample flow and the short axis (d ₁) of the elliptical spot is along the direction of the sample flow. In various embodiments, the ratio of d ₂︰d₁ is greater than 1 or in the range of about 2-5, 5-10, 10-15, 15-20, or 20-25.

In various embodiments, the major axis of the elliptical spot (d ₂) is greater than the width of the flow cell (d ₃). In various embodiments, the ratio of d ₂︰d₃ is in the range of about 2-5, 5-10, 10-15, 15-20, or 20-25.

In various embodiments, the elliptical spot on the flow cell has a diameter of about 4-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-99, or 99-100 μm in a direction parallel to the sample flow and a diameter of about 40-100, 100-500, 500-1000, 1000-1500, 1500-2000, 2000-2500, 2500-3000, 3000-3500, 3500-4000, 4000-4500, or 4500-5000 μm in a direction perpendicular to the sample flow. In some embodiments, the elliptical spot on the flow cell has a diameter of about 15-16, 16-20, 20-30, 30-40, or 40-50 μm in a direction parallel to the sample flow and a diameter of about 150-160, 160-200, 200-500, 500-1000, 1000-1500, 1500-2000, or 2000-2500 μm in a direction perpendicular to the sample flow.

In various embodiments, the sample flow formed within the flow chamber has a width in a direction perpendicular to the sample flow of about 4-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 μm. In some embodiments, the sample flow formed within the flow chamber has a width in a direction perpendicular to the sample flow of about 20-30, 30-40, or 40-50 μm.

In various embodiments, the flow chamber has a width in a direction perpendicular to the sample flow of about 4-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 μm. In some embodiments, the flow chamber has a width in a direction perpendicular to the sample flow of about 20-30, 30-40, or 40-50 μm. In various embodiments, the flow chamber has a width in the range of about 1-10, 10-40, 40-100, or 100-200 μm; and a depth in the range of about 1-10, 10-40, 40-100, or 100-200 μm. In various embodiments, the flow chamber has a length ranging from about 1-10, 10-100, 100-1000, 1000-5000, or 5000-10000 μm.

In some embodiments, the flow chamber is configured to form a sample flow without a sheath flow. In other embodiments, the flow chamber is configured as a sample stream formed by a sheath flow. In some embodiments, the sample stream is sheath-free. In other embodiments, the sample stream is surrounded by a sheath stream.

In various embodiments, the flow chamber includes a surface illuminated by the illumination light, and the surface is positioned non-perpendicular to a central axis of the illumination light. In some embodiments, the angle between the surface and the central axis of the illuminating light may be about 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-89, or 89-89.9 degrees.

In various embodiments, the flow chamber is part of a cartridge device configured to be placed in a reader for analysis, and the reader includes a light source, a condenser lens, a detector, and a signal analysis unit. In various embodiments, the cartridge device includes a surface illuminated by the illumination light and the surface is positioned non-perpendicular to a central axis of the illumination light. In some embodiments, the angle between the surface and the central axis of the illuminating light may be about 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-89, or 89-89.9 degrees.

In various embodiments, the flow cell or cartridge device housing the flow cell is positioned or tilted to an orientation in which the reflective surface of the flow cell or cartridge device is non-perpendicular to the central axis of the illumination light and directs the reflected light away from the light source. In certain embodiments, the angle between such reflective surface and the central axis of the illuminating light may be about 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-89, or 89-89.9 degrees.

In some embodiments, one lens in the focusing module is positioned or tilted to an orientation in which the reflecting surface of the lens is non-perpendicular to the central axis of the illuminating light and directs the reflected light away from the light source. In certain embodiments, the angle between such reflective surface and the central axis of the illuminating light may be about 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-89, or 89-89.9 degrees.

In various embodiments, the cartridge device is configured to mix the sample with the reagent to form a measurement sample and form a sample stream of the measurement sample within the flow chamber. In various embodiments, the method as described herein further comprises mixing the sample with the reagent using the cartridge device to form a measurement sample and forming a sample stream of the measurement sample within the flow chamber. In various embodiments, the reagent comprises a fluorescent labeling compound, an osmolality adjusting compound, a spheronizing compound, or a lysing compound, or a combination of a fluorescent labeling compound, an osmolality adjusting compound, a spheronizing compound, and a lysing compound. Non-limiting examples of reagents can be found in this disclosure and U.S. patent application Ser. No. 15/819,416, the disclosures of which are incorporated herein by reference in their entirety as if fully set forth.

In various embodiments, the reagent comprises a fluorescent marker compound. In various embodiments, the fluorescent-labeled compound comprises an antibody that binds to a fluorescent group, an antibody that binds to a fluorescent particle, or a fluorescent dye, or a combination of an antibody that binds to a fluorescent group, an antibody that binds to a fluorescent particle, and a fluorescent dye. In various embodiments, the reagent comprises a fluorescent dye.

In various embodiments, an apparatus or apparatus system as described herein further comprises a receiving module.

In various embodiments, the receiving module is configured to split the scattered light with forward angle and the fluorescence collected by the condensing lens into two separate light paths towards two separate detectors, respectively. In various embodiments, the receiving module is configured to split the scattered light with a lateral angle and the fluorescence collected by the condenser lens into two separate light paths towards two separate detectors, respectively. In various embodiments, the receiving module comprises a beam splitter, a dichroic mirror, a prism, or a diffraction grating, or a combination of a beam splitter, a dichroic mirror, a prism, and a diffraction grating. In some embodiments, the receiving module includes a doublet configured to focus the collected fluorescence light.

In various embodiments, an apparatus or apparatus system as described herein further comprises a doublet lens configured to focus the concentrated fluorescence light.

In various embodiments, the device or device system as described herein further comprises a signal analysis unit configured to analyze the signal of scattered light and the signal of fluorescence with a forward angle to analyze the particles and/or cells.

In various embodiments, the measurement sample comprises blood cells. In various embodiments, the sample comprises blood cells.

In various embodiments, analyzing the particles and/or cells includes analyzing blood cells. In various embodiments, analyzing the blood cells includes one or more of: measuring the number and/or percentage of leukocytes, identifying leukocytes as subtypes (e.g., lymphocytes, monocytes, neutrophils, eosinophils, and basophils), measuring the number and/or percentage of one subtype of leukocytes, measuring the number and/or percentage of erythrocytes, and measuring the number and/or percentage of platelets. In some embodiments, analyzing the blood cells includes measuring a number of red blood cells and a number of platelets.

In various embodiments, as shown in fig. 1A, a device or device system as disclosed herein includes a sample supply unit, a detection unit, and a signal analysis unit. The sample supply unit provides a measurement sample comprising cells or particles or both to the detection unit. In various embodiments, as shown in fig. 1B, the detection unit includes a light source, a focusing module, a flow cell, a condensing lens, a receiving module, and a detector. The sample supply unit supplies a measurement sample to the flow chamber of the detection unit. The sample is measured through the flow chamber to form a sample stream. Light emitted from the light source is used to illuminate the sample stream and an optical signal (e.g., scattered light with a forward angle, fluorescence, or both) from the sample stream is measured by the detector. Light emitted from the light source is shaped by the focusing module into a desired spot prior to illuminating the sample stream. The optical signal from the sample stream is collected by a collection lens and directed to a detector by a receiving module before being measured by the detector. The signal analysis unit analyzes the optical signals measured by the detector to obtain desired results (e.g., number of cells, characteristics of individual cells, and distribution and characterization of cell populations in the measurement sample). The receiving module may be used for various other functions: dividing the collected light into a plurality of light paths, filtering certain wavelengths of light from the collected light, focusing the collected light on a target point having a particular size or shape, or performing other light processing functions. In various embodiments, one, two, or more detectors may be used to measure one, two, or more types of optical signals from the sample stream.

Fig. 2A (side view) and 2B (top view) show one non-limiting example of a detection unit in which a flow cell 201 is used to form a sample flow of a measurement sample. The light source 202 emits illumination light to illuminate the sample stream. The focusing module 203 focuses the illumination light on the sample stream and shapes it into a specific beam shape. The condenser lens 204 condenses both scattered light with a forward angle and fluorescence from the sample stream illuminated by the illumination light. The receiving module 205 divides the collected light into two light paths. Two detectors, detector 206 and detector 207, detect and measure the collected light on two light paths. Beam blocker 208 may be positioned in the optical path between the flow cell and one of the detectors and block illumination light passing through the flow cell.

In this non-limiting example, the focusing module 203 includes a beam focusing lens 209, and two cylindrical lenses 210 and 211. The irradiation light emitted from the light source 201 is collimated by the beam converging lens 209 and forms parallel light having a circular beam shape or an elliptical beam shape. The two cylindrical lenses are positioned in such an orientation that the cylindrical axis of lens 210 and the cylindrical axis of lens 211 are perpendicular to each other. The flow cell 201 is positioned at or near the focal point of the lens 211 and away from the focal point of the lens 210, wherein the illumination light is focused and shaped into a beam having an elliptical shape on the sample stream.

The condenser lens 204 is a beam focusing lens and the flow cell 201 may be positioned at the focal point of the condenser lens 204 or near the focal point of the condenser lens 204. The signal from the measurement sample is collected and beamed by the condenser lens 204 before entering the receiving module 205. The collected signals include, but are not limited to, scattered light from the sample stream having a forward angle and fluorescence. The signal from the measurement sample is collimated into parallel light by the condenser lens 204. In addition, a beam blocker 208 may be positioned behind the condenser lens 204 to block the illumination light from entering the receiving module.

The receiving module 205 includes a dichroic mirror 212, and the dichroic mirror 212 is inclined at an angle of 45 degrees with respect to the collected parallel light. The dichroic mirror 212 is a long-pass dichroic mirror that reflects light having a wavelength less than a specified threshold. Scattered light from the sample stream has the same or close wavelength as the illuminating light. The fluorescence from the sample stream has a wavelength spectrum that includes wavelengths greater than the wavelength of the illuminating light. By selecting a dichroic mirror having a threshold wavelength greater than the illumination light but less than the desired fluorescence, it splits the scattered light and fluorescence into two light paths. Alternatively, the receiving module may use other optical configurations (e.g., a beam splitter, a combination of beam splitters and optical filters, a prism, a diffraction grating, etc.) to split into scattered light and fluorescence. In this example, a beam focusing lens 213 is positioned in front of the detector 206 and focuses light passing through the dichroic mirror onto the detector. The light reflected by the dichroic mirror is received by the detector 207. An aperture 214 made of an opaque material and having a transparent opening in the center may be positioned to be in front of the detector 207. The aperture 214 blocks light outside the transparent opening from entering the detector 207.

A condenser lens 204 in the detection unit is used to collect both scattered light with a forward angle from the sample stream as well as fluorescence. In some embodiments, the concentrated scattered light comprises light having a scatter angle of less than about 20 degrees. In some embodiments, the concentrated scattered light comprises light having a scatter angle of less than about 15 degrees. In some embodiments, the concentrated scattered light comprises light having a scatter angle of less than about 10 degrees. In some embodiments, the concentrated scattered light comprises light having a scatter angle of less than about 5 degrees. In some embodiments, the concentrated scattered light comprises light having a scatter angle of less than about 4 degrees. In some embodiments, the concentrated scattered light comprises light having a scatter angle of less than about 3 degrees. In some embodiments, the concentrated scattered light includes light from elastic scattering. In some embodiments, the concentrated scattered light includes light from inelastic scattering. In various embodiments, the condenser lens is a spherical lens, an aspherical lens, or a doublet, or a combination of a spherical lens, an aspherical lens, and a doublet.

In a conventional analyzer such as that described in U.S. patent 7580120, scattered light having a forward angle and fluorescence are not collected in the same condenser lens; in contrast, scattered light having a forward angle is collected in one condenser lens and fluorescence is collected in the other condenser lens. The lens that collects the fluorescence is positioned perpendicular to the direction of the illuminating light, and therefore collects only scattered light having a lateral angle, which typically has a scattering angle greater than about 70 degrees. This configuration requires that the flow cell has at least two optically transparent surfaces, one for collection of scattered light with a forward angle and the other for collection of fluorescent signals.

In contrast, by focusing the fluorescence and scattered light with a forward angle in the same condenser lens, one optically transparent surface is sufficient for detection of both signals. In low cost flow cells, such as those constructed using plastic injection molding, it is more cost effective to have one optically transparent surface than to have two optically transparent surfaces.

The intensity of the fluorescent and scattered light may be significantly lower than the intensity of the illuminating light. In order to detect signals with a satisfactory signal-to-noise ratio (SNR), it is therefore desirable to remove the illuminating light from the concentrated light. In the non-limiting example of fig. 2A and 2B, a beam blocker 208 is used to block light located behind the flow chamber. When blocking the irradiation light, as shown in fig. 3A, the beam blocker also blocks the scattered light having a scattering angle smaller than θ ₁. The value of θ ₁ is adjusted by both the size and shape of the beam blocker and the distance of the beam blocker from the sample stream. As a non-limiting example shown in fig. 3B, the beam blocker may be a light blocking strip comprising an opaque material. The size of the light blocking strip is specified to be larger than the spot of the illuminating light at the beam blocker. In some embodiments, the beam blocker has a surface that minimizes reflection of the illuminating light to reduce stray light in the detection unit. Examples of such surfaces include, but are not limited to, surfaces made of light absorbing materials (e.g., black paint and anodized aluminum), and the like.

A beam blocker is positioned in the optical path between the flow cell and one of the detectors to block illumination light. In the non-limiting example of fig. 4A, the beam blocker is positioned between the flow chamber and the condenser lens. In the non-limiting example of fig. 4B, the beam blocker is positioned behind the condenser lens.

In some embodiments it is advantageous to position the beam blocker to be located behind the condenser lens. In order to collect fluorescence from the sample stream, the condensing lens is preferably close in distance to the flow cell, thereby enlarging the collection angle of the fluorescence. If a beam blocker is positioned between the flow cell and the condenser lens, the distance from the sample flow to the beam blocker is limited and a beam blocker of a given size can block scattered light having a large θ ₁. By positioning the beam blocker to be behind the condenser lens, it increases the distance from the sample to the beam blocker without sacrificing the distance from the sample to the condenser lens. Thus, for a beam blocker of a given size, the angle θ ₁ can be reduced. In some embodiments, the condensing lens itself may have a thickness in the range of 5-15 mm. The lens thickness may help to increase the substantial distance between the sample stream and the beam blocker.

In some embodiments, a spherical lens is used as a condensing lens to collect fluorescence from the sample stream and scattered light with a forward angle. As shown in fig. 5A, the spherical lens has at least one curved surface having a spherical shape. In some embodiments, a spherical lens is used as a condensing lens to collect fluorescence and scattered light with a forward angle. The aspheric lens facilitates increasing the collection efficiency of fluorescence from the sample stream. As shown in fig. 5B, the aspherical lens has at least one curved surface having an aspherical shape defined by the following equation:

X: a position in the optical axis direction;

Y: a distance from the center of the lens in a direction in which the optical axis advances;

K: a shape factor;

c ₀: a coefficient representing the curvature of the surface of the substrate (aspherical spherical substrate);

C _i: aspheric coefficients; and

I: integers (1 to n).

In the detection unit of fig. 2A and 2B, an elliptical spot is used to illuminate the sample flow in the flow chamber. The elliptical spot is obtained by using two cylindrical lenses 210 and 211. The two cylindrical lenses are positioned in such an orientation that their cylindrical axes are perpendicular to each other. As shown in fig. 6A, the flow cell is positioned at the focal point of the lens 211 or near the focal point of the lens 211. Thus, the diameter (D ₁) of the elliptical spot in the sample flow direction, i.e., the diameter in the y-axis direction as shown in fig. 6C and 6D, is narrow. At the same time, as shown in fig. 6B, the flow cell is positioned away from the focal point of the lens 210. Thus, the diameter (D ₂) of the elliptical spot perpendicular to the sample flow direction is wide, i.e., the diameter along the x-axis direction as shown in fig. 6C and 6D. The aspect ratio R of an elliptical spot is defined as:

R＝d₂/d₁。

The elliptical spot has an aspect ratio of R > 1. When r=1, the spot becomes circular. As shown in fig. 6D, the width of the sample stream illuminated by the spot is denoted as D ₃. One benefit of using an elliptical spot is that the two diameters d ₁ and d ₂ can be optimized separately. In various embodiments, d ₁ and d ₂ may be adjusted in a device or device system as described herein by adjusting the configuration of the focus module, e.g., adjusting the position of the light source, adjusting the positions of the two cylindrical lenses 210 and 211, and adjusting the position of the flow chamber, or by replacing these components.

For example, the diameter d ₁ can be optimized to measure cells of different sizes. To fully irradiate the cells with the spot, the diameter d ₁ should be larger than the diameter of the target cells. However, to reduce the background illuminated by the spot, the diameter d ₁ should be as small as possible. Thus, the diameter d ₁ is often selected to be close to or slightly larger than the diameter of the target cell. This helps to increase the amplitude of the fluorescence from the measured sample and helps to improve the signal to noise ratio of the fluorescence from the target cells.

In some embodiments, d ₁ of about 4-7 μm is used to measure cells having a diameter of about 1-3 μm. In some embodiments, d ₁ of about 7-10 μm is used to measure cells having a diameter of about 1-6 μm. In some embodiments, d ₁ of about 10-20 μm is used to measure cells having a diameter of about 1-9 μm. In some embodiments, d ₁ of about 20-30 μm is used to measure cells having a diameter of about 1-19 μm. In some embodiments, d ₁ of about 30-50 μm is used to measure cells having a diameter of about 1-29 μm. In some embodiments, d ₁ of about 50-80 μm is used to measure cells having a diameter of about 1-49 μm. In some embodiments, d ₁ of about 80-99 μm is used to measure cells having a diameter of about 1-79 μm.

For another example, the diameter d ₂ may be optimized for alignment between the sample flow and the illumination light in the spot. Some conventional analyzers, such as those disclosed in U.S. patent application 2015/0309049A1, use a circular light spot to illuminate a sample stream. The circular spot has an aspect ratio of r=1 (i.e. d ₂＝d₁). After d ₁ is selected for cell size, the circular spot limits the selection of d ₂. Fig. 7A shows one non-limiting example of a circular spot being used to illuminate the sample stream. As shown in fig. 7B, when the spot has an alignment deviation Δx from the sample stream, the spot is no longer able to illuminate the sample stream. For example, when the spot has a d ₁ of 20 μm, a circular spot limits d ₂ to equal d ₁. For a sample stream width d ₃ of 20 μm, a misalignment of 20 μm will result in the spot not illuminating the sample stream.

In contrast, both diameters d ₁ and d ₂ in an elliptical spot can be optimized separately. The elliptical spot has an aspect ratio of R >1 (i.e., d ₂>d₁). Fig. 7C shows one non-limiting example where an elliptical spot is used to illuminate the sample stream. As shown in fig. 7D, the spot can still illuminate the sample stream with the same amount of alignment deviation Δx. For example, when the spot has a d ₁ of 20 μm, the elliptical spot may still have a d ₂ greater than d ₁, e.g., 200 μm. For a 20 μm sample stream width d ₃, the spot can illuminate the sample stream even with a 20 μm misalignment. The tolerance of the misalignment between the flow cell and the spot is particularly important for applications where the flow cell is replaceable or disposable after measurement. In addition, illumination light with a gaussian beam profile is often used in cytometry analysis, and the wide d ₂ also helps to improve the uniformity of the illumination light across the width of the sample stream.

In some embodiments, d ₂ of about 40-500 μm is used to illuminate a flow cell having a sample flow width of about 4-10 μm. In some embodiments, d ₂ of about 100-1000 μm is used to illuminate a flow cell having a sample flow width of about 10-20 μm. In some embodiments, d ₂ of about 200-1500 μm is used to illuminate a flow cell having a sample flow width of about 20-30 μm. In some embodiments, d ₂ of about 300-2000 μm is used to illuminate a flow cell having a sample flow width of about 30-40 μm. In some embodiments, d ₂ of about 400-2500 μm is used to illuminate a flow cell having a sample flow width of about 40-50 μm. In some embodiments, d ₂ of about 500-5000 μm is used to illuminate a flow cell having a sample flow width of about 50-100 μm.

In the example of fig. 2A and 2B, an aperture 214 is used to block light outside the transparent opening of the aperture from entering the detector 207. Fig. 8 is an enlarged view of the detection unit of fig. 2B, as shown in fig. 8, by positioning the transparent opening of the aperture 214 to be located at the center of the detector 207 and adjusting the size of the opening, the aperture can be used to block scattered light having a scattering angle greater than the threshold angle θ ₂.

In the example of fig. 2A and 2B, an elliptical spot is obtained by focusing light emitted from a light source with a focusing module 203 comprising two cylindrical lenses 210 and 211. In other embodiments, elliptical spots may be obtained by using other focusing module configurations, including, but not limited to, modules with only one cylindrical lens, modules without a cylindrical lens, modules with anamorphic prism pairs (e.g., U.S. patent No. 5596456, the entire contents of which are incorporated herein by reference as if fully set forth), modules with diffraction grating components, and modules with other beam shaping optics (e.g., U.S. patent No. 6975458, the entire contents of which are incorporated herein by reference as if fully set forth), and so forth.

Fig. 9A (side view) and 9B (top view) illustrate another non-limiting example of a method for obtaining an elliptical spot, wherein the focusing module 903 comprises a beam focusing lens 909 and a cylindrical lens 911. Light emitted from the light source 902 is collimated into parallel light by the beam-condensing lens 909, and further passes through the cylindrical lens 911. Along the cylindrical axis of the lens 911, the beam is focused by the curved surface of the cylindrical lens (i.e., focused along the y-axis herein). The beam perpendicular to the cylindrical axis of lens 911 is not focused and remains parallel. The illumination light forms an elliptical spot on the sample stream by the flow cell being positioned at or near the focal point of the cylindrical lens 901. In various embodiments, d ₁ and d ₂ may be adjusted in an apparatus or apparatus system as described herein by adjusting the focus module configuration, e.g., adjusting the position of the light source, adjusting the position of the beam focusing lens 909, adjusting the position of the cylindrical lens 911, and adjusting the position of the flow cell, or replacing these components.

In this example, a condenser lens 904 is used to collect both fluorescence from the sample stream and scattered light having a forward angle. The beam blocker 908 is positioned to be located behind the condenser lens to block the illuminating light from entering the receiving module 905. The receiving module 905 includes a dichroic mirror 912, and the dichroic mirror 912 divides the scattered light having a forward angle and the fluorescent light into two optical paths. The fluorescence light passing through the dichroic mirror by the beam focusing lens 913 is focused on the detector 906. An aperture 914 located in front of detector 907 limits the scattered light that can be received onto detector 907.

The detection unit may use any type of flow cell design including, but not limited to, a sheathed flow cell, a non-sheathed flow cell, and the like. In some embodiments, as shown in fig. 10A, a flow cell with a sheath flow may be used, wherein the sample flow is surrounded by the sheath flow within the flow cell. The sheath flow focuses the sample flow to include a stream (stream) having a width smaller than the inner width of the flow chamber in a direction perpendicular to the sample flow. In some embodiments, as shown in fig. 10B, a flow cell without sheath flow may be used. In this sheath-less flow cell design, the sample flow is limited by the physical geometry of the flow cell and has a width in the direction perpendicular to the sample flow equal to the inner width of the flow cell. In certain embodiments, the detection unit uses a sheath-less flow cell. Examples of sheath-less flow chambers include, but are not limited to, those disclosed in U.S. patent application Ser. No. 62/497075, U.S. patent application Ser. No. 15/803133, and U.S. patent application Ser. No. 15/819416, the disclosures of which are incorporated herein by reference in their entirety as if fully set forth.

The detection unit may use any type of light source to provide illumination light that illuminates the sample stream, including but not limited to laser modules, laser diodes, LED devices, halogen lamps, and the like. In some embodiments, as shown in fig. 11, the light source includes a light emitting component, an optical fiber, and a beam focusing lens. The light emitting part emits light focused on one end of the optical fiber by the beam focusing lens. Light exiting from the other end of the fiber is used to illuminate the sample stream. In certain embodiments, the optical fiber is a single mode optical fiber. The use of single mode optical fibers may be advantageous. For example, when multimode light enters a single mode fiber, some components of the multimode light are removed by the single mode fiber and the light exiting the single mode fiber becomes single mode (e.g., substantially gaussian mode). In certain embodiments, the light emitting component is a laser diode, an LED device, or a halogen lamp.

The detection unit of the flow cytometer may use any type of photodetector to measure the signals of fluorescence and scattered light, including, but not limited to, bipolar phototransistors, photosensitive field effect transistors, photomultiplier tubes (PMTs), avalanche Photodiodes (APDs), photodiodes, CCD devices, CMOS devices, silicon photomultipliers (sipms), and the like. In some embodiments, the detection unit measures the intensity of the optical signal. In some embodiments, the detection unit measures the duration of the optical signal. In some embodiments, the detection unit measures the spatial distribution of the optical signal. In some embodiments, the detection unit measures image information of the optical signal.

In various embodiments, a signal analysis unit is used to analyze signals measured by the detector of the detection unit. In some embodiments, the signal analysis unit analyzes the signal of scattered light and the signal of fluorescence from the detection unit to measure particles and/or cells in the sample stream.

In various embodiments, the flow chamber is part of a cartridge device. Non-limiting examples of cartridge devices having flow chambers are shown in U.S. patent application Ser. No. 15/803133 and U.S. patent application Ser. No. 15/819416, the disclosures of which are incorporated herein by reference in their entirety as if fully set forth herein. In various embodiments, the cartridge device is placed in an analyzer device that includes a light source, a condenser lens, and a detector to measure particles and/or cells of a sample stream within a flow chamber. In various embodiments, the cartridge device is removed from the analyzer device after the measurement is completed. In some embodiments, the cartridge device receives a sample having particles and/or cells and further prepares a measurement sample from the sample having particles and/or cells and a reagent, and then provides the measurement sample to the flow chamber to form a sample stream for measurement.

The device or device system as described herein may be used to analyze any type of sample comprising cells. It can also be used to analyze any type of sample containing particles including, but not limited to, droplets, molecules (e.g., nucleic acid molecules, protein molecules, etc.) viruses, beads, nanoparticles, and the like. Its sample supply unit provides its detection unit with a measurement sample containing cells, particles or both. Its detection unit detects various signals from cells, particles or both in the measurement sample. Its analysis unit analyzes the detected signal (e.g., scattered light with a forward angle, fluorescence, or both) to obtain information about the measured sample (e.g., cell number, intrinsic fluorescence of an individual cell, fluorescent lambkin of an individual cell labeled with a fluorescent group). Based on the detected signals, the analysis unit may further obtain additional information of the measurement sample, e.g. classifying the cells into different types, characterizing individual cells, characterizing cell populations in the measurement sample, etc.

In some embodiments, the devices or device systems described herein are used to analyze cells in a blood sample (e.g., a blood sample from a human or other species such as canine, feline, equine, bovine, ferret, gerbil, rabbit, porcine, mini-porcine, guinea pig, etc.). As a non-limiting example, a device or device system may be used to analyze a human blood sample to detect and classify cells in the human blood sample into three main types including white blood cells, red blood cells, and platelets. The device or device system may also be used to divide leukocytes into five major subtypes including lymphocytes, monocytes, neutrophils, eosinophils, and basophils. The device or device system may also be used to detect the presence and level of antigen expression by cells and use the antigen expression level to classify cells into different types. For one non-limiting example, a device or device system may be used to divide lymphocytes into T cells, NK cells, CD4 ⁺ cells, CD8 ⁺ cells, and the like. The device or device system may be used to further detect and classify other cells in a human blood sample, for example, those cells as described in U.S. patent application No. 2014/0170680A1, the entire contents of which are incorporated herein by reference as if fully set forth herein.

For any type of biological sample for measurement, the cells in the sample have a known size range. The size of the sample flow in the flow chamber and the diameter of the elliptical spot can thus be optimized accordingly in the detection unit. For example, cells in a human blood sample may be analyzed. Human blood cells have a known size range, for example, platelet cells having a diameter of about 1-3 μm, red blood cells having a diameter of about 6-8 μm, and white blood cells having a diameter of about 7-15 μm. Accordingly, the detection unit may use a sample stream having a d ₃ of about 20-50 μm, and an elliptical spot having a d ₁ of about 16-50 μm and a d ₂ of about 160-2500 μm.

In some embodiments, a device or device system as described herein is used to analyze white blood cells in a blood sample. In some embodiments, the sample supply unit prepares the measurement sample by mixing the blood sample with a staining reagent containing at least a fluorescent labeling compound, including but not limited to an antibody that binds to a fluorescent group, an antibody that binds to a fluorescent particle, a fluorescent dye, and the like. Fluorescent labeling compounds label leukocytes with high affinity.

In some embodiments, the sample supply unit prepares the measurement sample by mixing the blood sample with a staining reagent containing at least a fluorescent dye. The fluorescent dye may be a nucleic acid dye. Examples of fluorescent dyes include, but are not limited TO, propidium iodide, ethidium bromide, DAPI, hoechst dye, acridine orange, 7-AAD, LDS751, TOTO dye family, TO-PRO dye family, SYTO dye family, thiazole orange, basic orange 21, auramine-O, and dye compounds, and the like, as disclosed in U.S. patent No. 6004816, the entire contents of which are incorporated herein by reference as if fully set forth. Fluorescent dyes label nucleic acids of leukocytes with high affinity.

The prepared measurement sample is supplied into the flow chamber of the detection unit to form a sample stream. The sample stream is illuminated by illumination light and the signal (e.g., fluorescence and scattered light with a forward angle) from the sample stream is measured by two detectors in the detection unit. The analysis unit analyzes the detected signals (e.g., intensities of fluorescent and scattered light) to obtain information of the measurement sample, including but not limited to one or more of the following parameters: total number of leukocytes, and counts and percentages of different subtypes (lymphocytes, monocytes, neutrophils, eosinophils, basophils, etc.) in leukocytes.

In some embodiments, the staining reagent further comprises a lysing compound that lyses erythrocytes in the blood sample. Since the concentration of erythrocytes is usually higher than the concentration of leukocytes, this helps to improve the detection of signals from leukocytes in the sample stream. Examples of lysing compounds include, but are not limited to, ammonium salts, quaternary ammonium salts, pyridinium salts, hydroxylamine salts, nonionic surfactants, ionic surfactants, sodium Dodecyl Sulfate (SDS), sodium Lauryl Sulfate (SLS), and combinations of ammonium salts, quaternary ammonium salts, pyridinium salts, hydroxylamine salts, nonionic surfactants, ionic surfactants, sodium Dodecyl Sulfate (SDS), sodium Lauryl Sulfate (SLS), and any other known erythrocyte lysing compound.

In one non-limiting example, the fluorescent dye in the coloring agent is thiazole orange. A dichroic mirror with a long pass threshold wavelength of 506nm is used to separate the fluorescent signal from the collected light. The blocking strip is used as a beam blocker to block the irradiation light. The barrier rib has a rib width that blocks scattered light having a scattering angle of θ ₁ of less than about 4 degrees. An aperture having a transparent opening is used in front of the detector, the aperture receiving light comprising scattered light, and the aperture blocking scattered light having a scatter angle of θ ₂ greater than about 12 degrees from entering the detector. The analysis unit uses the detected signals of fluorescence and scattered light to generate a scatter plot. In one non-limiting example of a scatter plot, as shown in FIG. 12, each dot represents white blood cells being detected in a sample stream. The analysis unit counts the total number of points in the scatter plot to determine a total count of white blood cells in the blood sample. Furthermore, the points in the scatter plot fall into different clusters. The number of spots in each cluster is analyzed in unit calendar to determine the counts and percentages of the different subtypes in leukocytes (including lymphocytes, monocytes, neutrophils, eosinophils, and basophils).

In another non-limiting example, the fluorescent dye in the coloring agent is acridine orange. A dichroic mirror with a long pass threshold wavelength of 610nm is used to separate the fluorescent signal from the collected light. The analysis unit generates a histogram using the signal of the detected fluorescence. In one non-limiting example of a histogram, as shown in fig. 13, fluorescence intensity is plotted as x-axis, and the number of cells detected with corresponding fluorescence intensity is plotted as y-axis. The histogram indicates three different peaks. Peaks with low fluorescence intensities correspond to lymphocytes; peaks with moderate fluorescence intensities correspond to monocytes; and the peak having high fluorescence intensity corresponds to granulocytes including neutrophils, eosinophils and basophils. The cell calendar number of cells in all peaks was analyzed to determine the total count of leukocytes, and the number of cells in each peak was also calendar to determine the counts and percentages of lymphocytes, monocytes and granulocytes.

In some embodiments, a device or device system as described herein is used to analyze red blood cells and platelets in a blood sample. In some embodiments, the sample supply unit prepares the measurement sample by mixing the blood sample with a staining reagent containing at least a fluorescent labeling compound, including but not limited to an antibody that binds to a fluorescent group, an antibody that binds to a fluorescent particle, a fluorescent dye, and the like. Fluorescent labeling compounds label erythrocytes and platelets with high affinity.

In some embodiments, the sample supply unit prepares the measurement sample by mixing the blood sample with a staining reagent containing at least a rongeur dye. The fluorescent dye may be a nucleic acid dye. Examples of nucleic acid dyes include, but are not limited TO, propidium iodide, ethidium bromide, DAPI, hoechst dye, acridine orange, 7-AAD, LDS751, TOTO dye family, TO-PRO dye family, SYTO dye family, thiazole orange, basic orange 21, auramine-O, and dye compounds, and the like, as disclosed in U.S. patent No. 6004816, the entire contents of which are incorporated herein by reference as if fully set forth. Fluorescent dyes label nucleic acids of erythrocytes and platelets with high affinity.

The prepared measurement sample is supplied to the flow cell of the detection unit to form a sample stream. The sample stream is illuminated by illumination light and signals from the sample stream (e.g., fluorescence and scattered light with forward angles) are measured by two detectors in the detection unit. The analysis unit analyzes the detected signals (e.g., intensities of fluorescent and scattered light) to obtain information of the measurement sample, including but not limited to one or more of the following parameters: total count of red blood cells, total count of platelets, size of individual red blood cells, size distribution of red blood cell population, size distribution of individual platelets, count of reticulocytes, and the like.

In some embodiments, the coloring agent further comprises a spheronizing compound. The spheronizing compound is used to transform erythrocytes in the prepared sample from a cake shape to a spherical shape. In the case of a sphere, the intensity of scattered light from a single red blood cell becomes independent of the orientation of the cells in the flow chamber. Examples of spheronizing compounds include, but are not limited to, surfactants such as Sodium Dodecyl Sulfate (SDS) and Sodium Lauryl Sulfate (SLS).

In one non-limiting example, the fluorescent dye used in the coloring agent is acridine orange. A dichroic mirror with a long pass threshold of 590nm is used to separate the fluorescent signal from the collected light. The blocking strip is used as a beam blocker to block the irradiation light. The blocking bars have a bar width that blocks scattered light having a scattering angle of θ ₁ of less than about 1 degree. An aperture having a transparent opening is used in front of the detector, the aperture receiving light comprising scattered light, and the aperture blocking scattered light having a scatter angle of θ ₂ greater than about 5 degrees from entering the detector. The analysis unit uses the detected signals of fluorescence and scattered light to generate a scatter plot. In one non-limiting example of a scatter plot, as shown in FIG. 14, each dot represents cells being detected in a sample stream. The scatter plot falls into two different clusters. Clusters with lower scattered light intensity and higher fluorescence intensity correspond to platelets, while clusters with higher scattered light intensity and lower fluorescence intensity correspond to red blood cells. The number of points in each cluster is analyzed to determine the total count of platelets and the total count of red blood cells. The analysis unit may evaluate the scattered light intensities of all points in the red blood cell cluster to determine the size of individual red blood cells and the size distribution of the red blood cell population in the measurement sample. The analysis unit may also evaluate the scattered light intensities of all points in the platelet cluster to determine the size of individual platelets and the size distribution of the platelet cluster in the measurement sample.

In some embodiments, a device or device system as described herein is used to analyze red blood cells and platelets in a blood sample. The sample supply unit prepares the measurement sample by mixing the blood sample with at least a diluting reagent containing an osmolarity adjusting agent for the prepared sample. The reagent is used to dilute the concentration of red blood cells in the prepared sample while minimizing unwanted lysis of the red blood cells. Examples of osmolarity adjusting compounds include, but are not limited to: salts comprising cations (e.g., salts comprising Na ⁺、K⁺、NH4⁺、Ca²⁺ and Mg ²⁺); salts comprising anions (e.g., ,Cl^-、Br^-、NO₃ ^-、CO₃ ^2-、HCO₃ ^-、SO₄ ^2-、HSO₄ ^-、PO₄ ^3-、HPO₄ ^2-、H₂PO₄ ^-、COOH^- and CH ₃COO^-); organic compounds such as sugars (e.g., glucose and sucrose); and alcohols (e.g., ethanol and methanol), etc. The prepared measurement sample is supplied to the flow cell of the detection unit to form a sample stream. The sample flow is illuminated by illumination light and a signal of scattered light with a forward angle is measured in a detector. The analysis unit analyzes the detected signals to obtain information of the measured sample including, but not limited to, one or more of the following parameters: total count of red blood cells, total count of platelets, size of individual red blood cells, size distribution of red blood cell population, size distribution of individual platelets, count of reticulocytes, and the like.

In one non-limiting example, the osmolarity adjusting compound in the diluent reagent is sodium chloride. In this example, a dichroic mirror may not be required. The blocking strip is used as a beam blocker to block the irradiation light. The blocking bars have a bar width that blocks scattered light having a scattering angle of θ ₁ of less than about 1 degree. An aperture having a transparent opening is used in front of the detector, the aperture receiving light comprising scattered light, and the aperture blocking scattered light having a scatter angle of θ ₂ greater than about 7 degrees from entering the detector. As shown in fig. 15, the analysis unit causes the detected scattered light signal to generate a scatter pattern. In this histogram, the scattered light intensity is plotted as the x-axis and the number of cells detected with the corresponding scattered light intensity is plotted as the y-axis. The histogram indicates two distinct peaks. Peaks with lower intensities correspond to platelets; and peaks with higher intensities correspond to erythrocytes. The number of cells in each peak is analyzed to determine the total count of platelets and the total count of red blood cells. The analysis unit may evaluate the intensity of scattered light from cells in the red blood cell peak to determine the size of individual red blood cells and the size distribution of the red blood cell population in the sample. The analysis unit may also evaluate the scattered light intensity from cells in the platelet peaks to determine the size of individual platelets and the size distribution of platelet populations in the sample.

In some embodiments, a device or device system as described herein is used to analyze white blood cells, red blood cells, and platelets in a blood sample. The sample supply unit prepares a measurement sample by mixing a portion of the blood sample with a first staining reagent comprising at least a first fluorescent dye. In addition, the sample supply unit prepares another measurement sample by mixing another portion of the blood sample with a second staining reagent comprising at least a second fluorescent dye. The first coloring agent and the second coloring agent may be the same or different. The first fluorescent dye and the second fluorescent dye may be the same or different. They may be nucleic acid dyes. Examples of fluorescent dyes include, but are not limited TO, propidium iodide, ethidium bromide, DAPI, hoechst dye, acridine orange, 7-AAD, LDS751, TOTO dye family, TO-PRO dye family, SYTO dye family, thiazole orange, basic orange 21, auramine-O, and dye compounds, and the like, as disclosed in U.S. patent No. 6004816, the entire contents of which are incorporated herein by reference as if fully set forth.

In the detection unit, a measurement sample is first supplied to the flow chamber to form a first sample stream; the first sample stream is illuminated by a first illumination light; and the signal (e.g., fluorescence and scattered light with forward angle) is measured by two detectors. After completion of the measurement of the first sample stream, another measurement sample is supplied to the flow chamber to form a second sample stream; the second sample stream is illuminated by a second illumination light; and the signal (e.g., fluorescence and scattered light with forward angle) is measured by two detectors. The first illumination light and the second illumination light may be the same or different.

The analysis unit analyzes the detected signals (e.g., fluorescence and scattered light) to obtain information of the measurement sample, including but not limited to one or more of the following parameters: total count of white blood cells, count and percentage of different subtypes of white blood cells (e.g., lymphocytes, monocytes, neutrophils, eosinophils, basophils, etc.), total count of red blood cells, total count of platelets, size of individual red blood cells, size distribution of red blood cell population, size of individual platelets, size distribution of platelet population, count of reticulocytes, and the like.

In some embodiments, a device or device system as described herein is used to analyze white blood cells, red blood cells, and platelets in a blood sample. The sample supply unit prepares a first measurement sample by mixing a portion of the blood sample with a first reagent. The sample supply unit also prepares a second measurement sample by mixing a portion of the first measurement sample with a second reagent. The first reagent at least comprises a fluorescent dye.

In the detection unit, a second measurement sample is first supplied to the flow cell to form a sample stream and two optical signals (e.g. fluorescence and scattered light with a forward angle) are measured by two detectors. The measured fluorescence and scattered light signals are used by a signal analysis unit to determine the count of red blood cells or the count of platelets or both. After measuring the second sample, the first measurement sample is then supplied to a flow cell to form a sample stream and two optical signals (e.g., fluorescence and scattered light with a forward angle) are measured by two detectors. The measured fluorescence and scattered light signals are used by the signal analysis unit to determine the count of leukocytes, the count of different subtypes of leukocytes (e.g., lymphocytes, monocytes, neutrophils, eosinophils, basophils, etc.), the percentage of different subtypes of leukocytes (e.g., lymphocytes, monocytes, neutrophils, eosinophils, basophils, etc.), and the like. In other embodiments, the first measurement sample may be measured in the flow chamber before the second measurement sample is measured in the flow chamber.

When a condensing lens is used to collect both fluorescent and scattered light, the efficiency of collecting the light signal is an important consideration. For example, fluorescence typically has low intensity and optimized aggregation efficiency is important for improving detection sensitivity and signal-to-noise ratio.

Fig. 16A (top view) and 16B (side view) show one non-limiting example of a detection unit. The measurement sample is formed into a sample stream within the flow chamber 1601. The laser diode 1602 is used as a light source. The focusing module includes an aspherical lens 1603, an aperture 1604, a spherical lens 1605, and a cylindrical lens 1606. The illumination light from the laser diode 1602 is first collimated into parallel light by an aspherical lens 1603 and further focused by a lens pair 1605 and 1606 into an elliptical spot on the flow cell. An aperture 1604 is used to define the diameter of the collimated light. The condenser lens 1608 is used to collect the signal light from the flow cell into collimated light. The detection module further comprises a beam blocker 1607 between the flow cell and the condenser lens to block the illumination light. The receiving module includes a beam splitter 1609, a focusing lens 1610, a filter 1611, a second focusing lens 1613, and an aperture 1614. The beam splitter 1609 splits the collimated light from the condenser lens into two optical paths. In one optical path, the signal light passes through a focusing lens 1610 and a filter 1611, and is then measured by a detector 1612. In the other optical path, the signal light passes through a focusing lens 1613 and an aperture 1614, and is then measured by a detector 1615. By selecting a long pass filter or a band pass filter as the filter 1611, the detector 1612 measures the intensity of fluorescence from the sample stream. In some embodiments, beam splitter 1609 is a dichroic mirror. In certain embodiments, the dichroic mirror has a passband that matches the fluorescence wavelength.

The choice of condenser lens 1608 and focusing lens 1610 is important to improve the collection efficiency of the fluorescence from the flow cell. First, the condenser lens 1608 limits the maximum amount of fluorescence that can be collected as collimated light, and the maximum amount is determined by the numerical aperture of the condenser lens. Next, the selection of the condenser lens 1608 and the focusing lens 1610 determines the focused spot size of the fluorescence reaching the detector 1612. When the focused spot size is larger than the effective detection area of the detector, the fluorescence is not measured by the detector in the portions outside the effective detection area. This reduces the detectable signal strength and requires a more sensitive detector. This problem is particularly important for detectors with small effective detection areas, such as photodiodes, avalanche diodes (APDs), and silicon photomultipliers (sipms). For example, to measure fluorescence using avalanche diodes (APDs) as detectors, aspheric lenses are used as condensing lenses (e.g., U.S. patent No. 7894047 and U.S. patent No. 7580120, the disclosures of which are incorporated herein by reference in their entirety as if fully set forth).

In one non-limiting example of a device or device system as described herein, a spherical lens is used as the condensing lens 1608 and a doublet lens is used as the focusing lens 1610 to achieve optimal condensing efficiency. The doublet is made of two single lenses paired together. Fig. 17 shows one non-limiting example of a doublet comprising a first simple lens 1701 and a second simple lens 1702 mated together at an interface surface. Doublets are typically used to reduce achromatism, which is the aberration between light of different wavelengths. Here, a doublet is used to increase the collection efficiency of fluorescence in a device or device system as described herein.

Fig. 18A and 18B show a non-limiting example showing the improvement in collection efficiency in the case of using a doublet. In fig. 18A, a spherical lens 1803 is used as a condensing lens to collect fluorescent signals 1802 from the flow cell 1801. Another spherical lens 1804 is used as a first focusing lens to focus the collected fluorescence light into a focused spot 1805 on a detector 1806. In contrast, as shown in fig. 18, a doublet 1807 is used as a first focusing lens, and the collected fluorescence is focused into a focused spot 1808. The focused spot 1808 of fig. 18B has a much smaller size than the focused spot 1805 of fig. 18A. Thus, a detector with a smaller effective detection area can be used for measurements with a doublet.

As shown in the non-limiting examples of fig. 16A and 16B, there are several surfaces on the optical path of the illumination light that can reflect a portion of the illumination light back to the light source. Fig. 16C is an enlarged view of the light source, focusing module and flow cell. In this configuration, each of the planar surface 1619 of the spherical lens 1605, the planar surface 1620 of the cylindrical lens 1606, and the planar surface 1621 of the flow chamber 1601 may reflect a portion of the illumination light 1617 back to the light source 1602. If the reflected light 1618 enters the light source, it may cause the intensity of the illuminating light to fluctuate. Some types of light sources, such as laser diodes, are particularly susceptible to interference from reflected light. This fluctuation problem of the light source, coupled with the fact that the surface of the replaceable or disposable flow cell, which is made of a low cost plastic material, can reflect a large amount of illumination light, can lead to inaccurate detection of particles and/or cells by the detection unit. Thus, preferably, reflection of the illuminating light is eliminated or minimized. For example, the reflected light may be directed away from the light source.

In some embodiments, the focusing module is configured to eliminate or minimize reflection of illumination light from a surface of a component in the focusing module, a surface of the flow chamber, or a surface of a cartridge device housing the flow chamber. Non-limiting examples of cartridge devices containing flow chambers are described in U.S. patent application Ser. No. 15/803133 and U.S. patent application Ser. No. 15/819416, the disclosures of which are incorporated herein by reference in their entirety as if fully set forth herein. The cartridge device is received into a reader for analysis. In some embodiments, the detection unit is a component of a reader instrument. As one non-limiting example, to reduce reflection of the illumination light, an anti-reflective coating may be applied on the surface of a component in the focusing module, on the surface of the flow cell, or on the surface of a cartridge device housing the flow cell.

In the configuration of fig. 16C, the optical axis of the spherical lens 1605 and the optical axis of the cylindrical lens 1606 are coaxial. They are also coaxial with the central axis 1616 of the illumination light emitted from the light source. In this configuration, the illumination light 1617 is reflected by the surface 1621 of the flow chamber 1601, and the reflected light 1618 is directed to the light source 1602.

In some embodiments, the focusing module is configured to direct reflection of the illumination light away from the light source or to block reflection of the illumination light into the light source.

In one non-limiting example, as shown in fig. 16D, the optical axis of the spherical lens 1605 and the optical axis of the cylindrical lens 1606 are coaxial. However, they are not coaxial with the central axis 1616 of the irradiation light emitted from the light source. Thus, the reflected light 1618 is directed away from the light source and blocked from entering the light source by the aperture 1604. Fig. 16E shows an overview of a detection unit with two lenses 1605 and 1606 coaxial with each other but not with the central axis 1616 of the light emitted from the light source 1602.

Fig. 16F shows another non-limiting example in which lens 1606 is coaxial with the central axis 1616 of the illumination light, but lens 1605 is not coaxial with the central axis 1616 of the illumination light. In this configuration, the surface of the flow cell or the surface of the cartridge device housing the flow cell may be directed away from the light source 1602 and blocked from entering the light source 1602 by the aperture 1604.

Other arrangements may also work if they include at least one lens that is not coaxial with the central axis of the illuminating light. For example, if the lens 1606 is not coaxial with the central axis 1616 of the illumination light, but the lens 1605 is coaxial with the central axis 1616 of the illumination light, the surface of the flow cell or the surface of the cartridge device housing the flow cell may also be directed away from the light source 1602 and blocked from entering the light source 1602 by the aperture 1604.

In various embodiments, one or more optical components in the focusing module are positioned not to be coaxial with a central axis of the illumination light emitted from the light source. In some embodiments, such non-coaxial optical components include cylindrical lenses, spherical lenses, or both. In some embodiments, the optical axis of such non-coaxial optical components is positioned within a range of about 0.01 to 0.1, 0.1 to 1, 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100mm away from the central axis of the illuminating light.

In various embodiments, an aperture is used in the focusing module to block reflected light from entering the light source. The size of the transparent area of the aperture needs to be large enough to define the diameter of the collimated illumination light and small enough to block the reflected illumination light. In some embodiments, the diameter of the transparent region of the aperture ranges from about 0.1 to 1, 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100mm.

In some embodiments, other configurations of the focusing module may also be used to direct reflected illumination light away from the light source. Fig. 16G shows a non-limiting example in which the flow chamber 1601 is tilted in such a way that the surface 1621 of the flow chamber is not perpendicular to the illumination light 1617. For example, the angle θ ₃ between the surface 1621 and the central axis 1616 of the illuminating light 1617 is not equal to 90 degrees and may be about 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-89, or 89-89.9 degrees. Thus, the reflected light 1618 is directed away from the light source 1602 and blocked from entering the light source 1602 by the aperture 1604. In various embodiments, the flow cell or flow cell-containing cartridge device is positioned or tilted to an orientation in which the reflective surface of the flow cell or flow cell-containing cartridge device is not perpendicular to the central axis of the illumination light and directs the reflected light away from the light source. In some embodiments, the lens in the focusing module is positioned or tilted to an orientation such that the surface of the lens is not perpendicular to the central axis of the reflected light and directs the reflected light away from the light source.

Various embodiments of the present disclosure provide an apparatus or apparatus system. The device or device system comprises: a flow chamber configured to form a sample stream of a measurement sample; a light source configured to emit illumination light for illuminating the sample stream; a condensing lens configured to condense both scattered light having a forward angle and fluorescence from the sample stream; a first light detector configured to detect the collected scattered light; a second light detector configured to detect the aggregated fluorescence.

Various embodiments of the present disclosure provide a device or device system for analyzing cells (e.g., blood cells). The device or device system comprises: a flow chamber configured to form a sample stream of a measurement sample comprising cells; a light source configured to emit illumination light for illuminating the sample stream; a condensing lens configured to condense both scattered light having a forward angle and fluorescence from the sample stream; a first light detector configured to detect the collected scattered light; a second light detector configured to detect the aggregated fluorescence. In various embodiments, the cells are blood cells. In some embodiments, the cells are white blood cells, red blood cells, or platelets, or a combination of white blood cells, red blood cells, and platelets. In some embodiments, the cell is a lymphocyte, monocyte, neutrophil, eosinophil, or basophil, or a combination of a lymphocyte, monocyte, neutrophil, eosinophil, and basophil. In various embodiments, the blood cells are labeled with a fluorescent dye. In various embodiments, the fluorescent dye is a nucleic acid dye.

In various embodiments, the illumination light forms an elliptical spot on the sample stream. In various embodiments, the long axis (d ₂) of the elliptical spot is perpendicular to the direction of the sample flow and the short axis (d ₁) of the elliptical spot is along the direction of the sample flow. In some embodiments, the d ₂︰d₁ ratio is greater than 1. In some embodiments, the d ₂︰d₁ ratio is about 2-5. In some embodiments, the d ₂︰d₁ ratio is about 5-10. In some embodiments, the d 2:d1 ratio is about 10-15. In some embodiments, the d ₂︰d₁ ratio is about 15-20. In some embodiments, the d ₂︰d₁ ratio is about 20-25. In some embodiments, the d ₂︰d₁ ratio is about 25-40. In various embodiments, d ₂ is about 5-10, 10-15, 15-20, 20-25, 25-40, or 40-60 times the sample flow width (d ₃).

In various embodiments, the device or device system as described herein further comprises a focusing module configured to shape the illumination light into an elliptical spot on the sample stream. In some embodiments, the focusing module includes a cylindrical lens. In other embodiments, the focusing module comprises two cylindrical lenses, wherein the two cylindrical lenses are positioned with their cylindrical axes perpendicular to each other. In other embodiments, the focusing module includes more than two cylindrical lenses. In various embodiments, the focusing module includes a cylindrical lens, anamorphic prism pair, or a diffraction grating component, or a combination of a cylindrical lens, anamorphic prism pair, and diffraction grating component.

In various embodiments, a condensing lens of the detection unit is used to condense both fluorescent and scattered light. In some embodiments, a condensing lens of the detection unit is used to collect both fluorescence and forward scattered light (i.e., scattered light having a forward angle (e.g., a scattering angle of less than about 25 degrees)). In some embodiments, a condensing lens of the detection unit is used to collect both fluorescence and side scattered light (i.e., scattered light having a side angle (e.g., a scattering angle greater than about 25 degrees)).

In various embodiments, the scattered light collected by the condenser lens comprises scattered light having a forward angle. In various embodiments, the scattered light collected by the condenser lens comprises scattered light having a scatter angle of less than about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 degrees. In various embodiments, the scattered light detected by the first detector comprises scattered light having a forward angle. In various embodiments, the scattered light detected by the first detector comprises scattered light having a scatter angle of less than about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 degrees. In some embodiments, the concentrated scattered light includes light from elastic scattering. In some embodiments, the concentrated scattered light includes light from inelastic scattering.

In various embodiments, the scattered light collected by the condenser lens comprises scattered light having a lateral angle. In various embodiments, the scattered light collected by the condenser lens comprises scattered light having a scatter angle of greater than about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 degrees. In various embodiments, the scattered light detected by the first detector comprises scattered light having a lateral angle. In various embodiments, the scattered light detected by the first detector comprises scattered light having a scatter angle of greater than about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 degrees. In some embodiments, the concentrated scattered light includes light from elastic scattering. In some embodiments, the concentrated scattered light includes light from inelastic scattering.

In various embodiments, the detection unit may measure both fluorescence and side scattered light to analyze particles and/or cells in the sample stream. One non-limiting example of such an analysis is disclosed in U.S. patent No. 7894047, which is incorporated herein by reference in its entirety as if fully set forth. In such an example, the analyzer uses a condenser lens to collect both fluorescence and lateral scatter angles, and further measures the fluorescence and lateral scatter angles to analyze white blood cells in the sample.

In various embodiments, the detection unit may measure both fluorescence and forward scattered light to analyze particles and/or cells in the sample stream. One non-limiting example of such an analysis is disclosed in U.S. patent No. 6004816, which is incorporated herein by reference in its entirety as if fully set forth.

In some embodiments, the condensing lens is a lens. In some embodiments, one condenser lens is configured to collect both scattered light from the sample stream having a forward angle and fluorescence. In various embodiments, the collected scattered light and the collected fluorescence are detected by two separate detectors. In various embodiments, the collected scattered light and the collected fluorescence light are separated into two separate light paths.

In various embodiments, the second detector comprises a bipolar photodiode, a photosensitive field effect transistor, a photomultiplier tube, an avalanche photodiode, a CCD device, or a CMOS device, or a combination of a bipolar photodiode, a photosensitive field effect transistor, a photomultiplier tube, an avalanche photodiode, a CCD device, and a CMOS device.

In various embodiments, an apparatus or apparatus system as described herein further comprises a receiving module configured to split the collected scattered light and the collected fluorescence light into two separate optical paths. In various embodiments, the receiving module includes a dichroic mirror for separating the collected scattered light and the collected fluorescence light into two separate optical paths. In some embodiments, the receiving module reflects the scattered light to a first light detector and transmits the fluorescent light to a second light detector. In other embodiments, the receiving module transmits scattered light to the first light detector and reflects fluorescent light to the second light detector.

In some embodiments, the sample stream illuminated by the spot is formed within a flow cell that is sheath-free (i.e., a sheath-free flow cell). Examples of sheath-less flow chambers include, but are not limited to, the sheath-less flow chambers disclosed in U.S. patent application Ser. No. 62/497075, U.S. patent application Ser. No. 15/803133, and U.S. patent application Ser. No. 15/819416, the disclosures of which are incorporated herein by reference in their entirety as if fully set forth herein. In other embodiments, the sample stream illuminated by the spot is formed in a sheath flow cell.

In some embodiments, the illumination light is a gaussian beam. In various embodiments, the light source comprises a laser diode, an LED device, or a halogen lamp, or a combination of a laser diode, an LED device, and a halogen lamp. In various embodiments, the light source comprises: a light emitting part configured to emit light; an optical fiber; and a beam focusing lens configured to focus light at one end of the optical fiber so that the light exits at the other end of the optical fiber. In various embodiments, the light emitting component comprises a laser diode, an LED device, or a halogen lamp, or a combination of a laser diode, an LED device, and a halogen lamp. In some embodiments, the optical fiber is a single mode optical fiber.

In various embodiments, the device or device system as described herein further comprises a beam blocker positioned between the sample flow and the condenser lens, wherein the beam blocker is configured to block the illumination light. In various embodiments, the apparatus or apparatus system as described herein further comprises a beam blocker positioned behind the condenser lens, wherein the beam blocker is configured to block the illumination light.

In various embodiments, the device or device system as described herein further comprises an aperture located in the optical path from the sample flow to the first light detector, wherein the aperture is configured to limit scattered light from entering the first light detector.

Various embodiments of the present disclosure provide a method. The method comprises the following steps: using the flow chamber to form a sample stream of the measurement sample; illuminating the sample stream with illumination light emitted from a light source; using a condensing lens to collect both scattered light from the sample stream having a forward angle and fluorescence; detecting the collected scattered light using a first light detector; and a second photodetector is used to detect the collected fluorescence. In some embodiments, the method is performed two or more times using the same flow chamber. In some embodiments, the condensing lens is a lens.

Various embodiments of the present disclosure provide a method. The method comprises the following steps: forming a first sample stream of a first measurement sample using the flow chamber; illuminating the first sample stream with illumination light emitted from the light source; using a condensing lens to collect both scattered light from the first sample stream having a forward angle and fluorescence; detecting the collected scattered light using a first light detector; detecting the aggregated fluorescence using a second light detector; forming a second sample stream of a second measurement sample using the same flow cell; illuminating the second sample stream with illumination light emitted from the light source; using a condensing lens to collect both scattered light from the second sample stream having a forward angle and fluorescence; detecting the collected scattered light using a first light detector; and a second photodetector is used to detect the collected fluorescence. In some embodiments, the condensing lens is a lens.

Various embodiments of the present disclosure provide a method for analyzing cells (e.g., blood cells). The method comprises the following steps: using the flow chamber to form a sample stream of a measurement sample comprising cells; illuminating the sample stream with illumination light emitted from a light source; using a condensing lens to collect both scattered light from the sample stream having a forward angle and fluorescence; detecting the collected scattered light using a first light detector; and a second photodetector is used to detect the collected fluorescence. In some embodiments, the condensing lens is a lens. In various embodiments, the cells are blood cells. In some embodiments, the cells are white blood cells, red blood cells, or platelets, or a combination of white blood cells, red blood cells, and platelets. In some embodiments, the cell is a lymphocyte, monocyte, neutrophil, eosinophil, or basophil, or a combination of a lymphocyte, monocyte, neutrophil, eosinophil, and basophil. In various embodiments, the blood cells are labeled with a fluorescent dye. In certain embodiments, the fluorescent dye is a nucleic acid dye. In some embodiments, the method is performed two or more times using the same flow chamber.

Various embodiments of the present disclosure provide a method for analyzing cells (e.g., blood cells). The method comprises the following steps: using the flow chamber to form a first sample stream comprising a first measurement sample of cells; illuminating the first sample stream with illumination light emitted from the light source; using a condensing lens to collect both scattered light from the first sample stream having a forward angle and fluorescence; detecting the collected scattered light using a first light detector; detecting the aggregated fluorescence using a second light detector; forming a second sample stream comprising a second measurement sample of cells using the same flow chamber; illuminating the second sample stream with illumination light emitted from the light source; using a condensing lens to collect both scattered light from the second sample stream having a forward angle and fluorescence; detecting the collected scattered light using a first light detector; and a second photodetector is used to detect the collected fluorescence. In some embodiments, the condensing lens is a lens.

In various embodiments, the cells in the first measurement sample are labeled with a fluorescent dye. In certain embodiments, the fluorescent dye is a nucleic acid dye. In various embodiments, the cells in the first measurement sample are blood cells. In some embodiments, the cells in the first measurement sample are white blood cells, red blood cells, or platelets, or a combination of white blood cells, red blood cells, and platelets. In some embodiments, the cells in the first measurement sample are lymphocytes, monocytes, neutrophils, eosinophils, or basophils, or a combination of lymphocytes, monocytes, neutrophils, eosinophils, and basophils. In various embodiments, the blood cells in the first measurement sample are labeled with a fluorescent dye. In various embodiments, the cells in the second measurement sample are labeled with a fluorescent dye. In certain embodiments, the fluorescent dye is a nucleic acid dye. In various embodiments, the cells in the second measurement sample are blood cells. In some embodiments, the cells in the second measurement sample are white blood cells, red blood cells, or platelets, or a combination of white blood cells, red blood cells, and platelets. In some embodiments, the cells in the second measurement sample are lymphocytes, monocytes, neutrophils, eosinophils, or basophils, or a combination of lymphocytes, monocytes, neutrophils, eosinophils, and basophils.

In some embodiments, the method as described herein further comprises preparing the measurement sample by mixing the sample with the first staining and/or diluting reagent.

In various embodiments, the methods as described herein further comprise analyzing cells (e.g., blood cells) in the measurement sample using the detected signals of scattered light and/or fluorescence.

In various embodiments, the method as described herein further comprises dividing the collected scattered light and the collected fluorescence light into two separate light paths.

In various embodiments, the method as described herein further comprises blocking the illumination light using a beam blocker located between the sample stream and the condenser lens. In various embodiments, the method as described herein further comprises blocking the illuminating light using a beam blocker located behind the condenser lens.

In various embodiments, the measurement sample comprises cells or particles, or a combination of cells and particles. Examples of cells include, but are not limited to, blood cells. Examples of particles include, but are not limited to, droplets, molecules (e.g., nucleic acid molecules, protein molecules, etc.), viruses, and beads. In some embodiments, the measurement sample comprises white blood cells, red blood cells, or platelets, or a combination of white blood cells, red blood cells, and platelets. In some embodiments, the measurement sample comprises lymphocytes, monocytes, neutrophils, eosinophils, or basophils, or a combination of lymphocytes, monocytes, neutrophils, eosinophils, and basophils. In some embodiments, the measurement sample includes a droplet, a molecule (e.g., a nucleic acid molecule, a protein molecule, etc.), a virus, or a bead, or a combination of a droplet, a molecule (e.g., a nucleic acid molecule, a protein molecule, etc.), a virus, and a bead. In various embodiments, the cells are labeled with a fluorescent dye. In various embodiments, the particles are either fluorescent or labeled with a fluorescent dye. In various embodiments, the methods as described herein further comprise labeling the cells with a fluorescent dye.

Various embodiments of the present disclosure provide an apparatus or apparatus system comprising: a flow chamber for forming a sample stream from a measurement sample; a light source for emitting illumination light to illuminate the sample flow within the flow chamber; a focusing module for focusing the illumination light into an elliptical spot on the flow cell; a condensing lens for condensing both fluorescence and scattered light from the measurement sample; a receiving module for dividing the collected light into at least two light paths, wherein light in one light path comprises scattered light and is detected by the first detector and light in the other light path comprises fluorescence and is detected by the second detector; an analysis unit that analyzes the signals from the first and/or second detectors to obtain information of the measurement sample; in various embodiments, the analyzed signal includes at least one of a signal detected by the first detector and a signal detected by the second detector. In various embodiments, the measurement sample is a blood sample.

The range of scattered light detected by the first detector may be selected by, for example, adjusting the configuration of the condenser lens (e.g., its size, its distance from the sample stream, and its orientation relative to the direction of the impinging light). In various embodiments, the condenser lens is positioned generally toward the direction of the scattered light. In various embodiments, the scattered light detected by the first detector comprises scattered light having a forward angle (i.e., forward angle scattered light). In various embodiments, the scattered light detected by the first detector comprises scattered light having a scatter angle of less than about 3 degrees. In various embodiments, the scattered light detected by the first detector comprises scattered light having a scatter angle of less than about 5 degrees. In various embodiments, the scattered light detected by the first detector comprises scattered light having a scatter angle of less than about 10 degrees. In various embodiments, the scattered light detected by the first detector comprises scattered light having a scatter angle of less than about 15 degrees. In various embodiments, the scattered light detected by the first detector comprises scattered light having a scatter angle of less than about 20 degrees.

In various embodiments, the elliptical spot on the flow cell has a diameter of about 4-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-99, or 99-100 μm in a direction parallel to the sample flow and a diameter of about 40-100, 100-500, 500-1000, 1000-1500, 1500-2000, 2000-2500, 2500-3000, 3000-3500, 3500-4000, 4000-4500, or 4500-5000 μm in a direction perpendicular to the sample flow. In various embodiments, the sample flow formed within the flow chamber has a width in a direction perpendicular to the sample flow of about 4-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 μm.

In some embodiments, the sample stream illuminated by the spot is formed in a flow cell without a sheath flow (i.e., a sheath-free flow cell). Examples of sheath-less flow chambers include, but are not limited to, those disclosed in U.S. patent application Ser. No. 62/497075, U.S. patent application Ser. No. 15/803133, and U.S. patent application Ser. No. 15/819416, the disclosures of which are incorporated herein by reference in their entirety as if fully set forth. In other embodiments, the sample stream illuminated by the spot is formed in a sheath flow cell.

In various embodiments, the focusing module includes at least one cylindrical lens. In various embodiments, the focusing module includes anamorphic prism pairs.

In various embodiments, the condenser lens is an aspherical lens. In various embodiments, the device or device system further comprises a beam blocker for blocking the illumination light. In various embodiments, the beam blocker is positioned on the optical path from the condenser lens to one of the two detectors. In various embodiments, the beam blocker is positioned on the optical path from the flow chamber to the condenser lens. In various embodiments, the beam blocker is a blocking strip comprising a light blocking material. In various embodiments, the barrier ribs have a stripe width that blocks the illuminating light and scattered light at a scattering angle of less than about 1,3, or 5.

In various embodiments, the light source comprises a laser diode, an LED device, or a halogen lamp. In various embodiments, the light source includes a light emitting component configured to emit light, an optical fiber, and a condenser configured to focus the light at one end of the optical fiber, whereby light emitted from the other end of the optical fiber can illuminate the measurement sample. In various embodiments, the light emitting component is a laser diode, an LED device, or a halogen lamp. In various embodiments, the detector for detecting fluorescence comprises a photodiode. In various embodiments, the illumination light is a gaussian beam.

In various embodiments, the device or device system as described herein further comprises a sample supply unit configured to prepare the measurement sample by mixing the sample with a staining and/or diluting reagent. In various embodiments, the diluent agent comprises at least one osmolarity adjusting compound. In various embodiments, the coloring agent comprises at least one fluorescent dye. In various embodiments, the fluorescent dye is a nucleic acid dye that selectively binds to nucleic acids. In various embodiments, the staining reagent further comprises a lysing compound that lyses the red blood cells. In various embodiments, the staining reagent further includes a spheronizing compound that is spheronized using red blood cells. In various embodiments, the diluent reagent further comprises a spheronizing compound that spheronizes the red blood cells. In various embodiments, the sample is a blood sample.

In various embodiments, a device or device system as described herein may be used to analyze a blood sample and obtain information about blood cells in the blood sample. In various embodiments, the analysis unit classifies leukocytes in the measurement sample into one or more subtypes, including, but not limited to, lymphocytes, monocytes, neutrophils, eosinophils, and basophils. In various embodiments, the analysis unit separates blood cells in the measurement sample into red blood cells and platelets.

Various embodiments of the present disclosure provide a method for analyzing a blood sample. It comprises the following steps: preparing at least one measurement sample by mixing a portion or all of the blood sample with a staining and/or diluting reagent; forming a sample stream of a measurement sample within the flow chamber; focusing illumination light from a light source into an elliptical spot on a sample stream within a flow chamber; using a condensing lens to collect fluorescence and scattered light from the measurement sample; dividing the collected light from the condensing lens into at least two light paths, wherein the light in one light path including scattered light is detected by a first detector and the light in the other light path including fluorescence is detected by a second detector; and analyzing the signal from the detector to obtain information for measuring blood cells in the sample. In various embodiments, the signal being analyzed includes at least one of a signal detected by a first detector and a signal detected by a second detector. In various embodiments, the scattered light detected by the first detector comprises scattered light having a forward angle (i.e., forward angle scattered light). In various embodiments, the scattered light detected by the first detector comprises scattered light having a scatter angle of less than about 3 degrees. In various embodiments, the scattered light detected by the first detector comprises scattered light having a scatter angle of less than about 5 degrees. In various embodiments, the scattered light detected by the first detector comprises scattered light having a scatter angle of less than about 10 degrees. In various embodiments, the scattered light detected by the first detector comprises scattered light having a scatter angle of less than about 15 degrees. In various embodiments, the scattered light detected by the first detector comprises scattered light having a scatter angle of less than about 20 degrees.

In various embodiments, the method further comprises separating the blood cells into one or more types including, but not limited to, white blood cells, red blood cells, and platelets. In some embodiments, the method further comprises separating the blood cells into red blood cells and platelets. In various embodiments, the method further comprises separating leukocytes in the measurement sample into one or more subtypes of lymphocytes, monocytes, neutrophils, eosinophils, and basophils. In various embodiments, erythrocytes, such as in a measurement sample prepared for analysis of leukocytes, are lysed. In various embodiments, the method further comprises preparing at least one measurement sample by mixing a portion or all of the blood sample with the lysing compound, whereby erythrocytes are lysed in the measurement sample. In various embodiments, the method further comprises preparing at least one measurement sample by mixing a portion or all of the blood sample with the sphering compound, whereby erythrocytes are sphered in the measurement sample.

In various embodiments, a method as described herein comprises: preparing a first measurement sample by mixing blood with a first staining and/or diluting reagent; forming a first sample flow of a first measurement sample within the flow chamber; analyzing the signal from the detector to obtain information of white blood cells in the first measurement sample; preparing a second measurement sample by mixing blood with a second staining and/or diluting reagent; forming a sample stream of a second measurement sample within the flow chamber; and analyzing the signal from the detector to obtain information of red blood cells and platelets in the first sample, wherein the first sample stream and the second sample stream are formed in the flow chamber, respectively. In various embodiments, the information of the white blood cells obtained includes at least one of the following parameters: the total number of leukocytes, the number of lymphocytes, the number of monocytes, the number of neutrophils, the number of eosinophils and the number of basophils. In various embodiments, the obtained information of erythrocytes and platelets includes at least one of the following parameters: the number of erythrocytes, the number of platelets, the size of individual erythrocytes, the size distribution of the population of erythrocytes, the size distribution of individual platelets, the size distribution of the population of platelets, the number of reticulocytes, and the like.

In accordance with the present disclosure, the terms "first" and "second" are used to denote an identity and not to indicate any temporal order.

Many variations and alternative elements have been disclosed in the embodiments of the disclosure. Additional variations and alternative elements will be apparent to those skilled in the art. In these variations, but not limited to, the fluidic units, components, and structures selected for use in the devices and methods of the present disclosure, as well as the samples that may be analyzed therewith. Various embodiments of the disclosure may specifically include or exclude any of these variations or elements.

In some embodiments, numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term "about". As one non-limiting example, those of ordinary skill in the art will generally consider a difference in value (increase or decrease) of no more than 10% as the term "about". Accordingly, in some embodiments, the numerical parameters set forth in the written specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the particular embodiment. In some embodiments, numerical parameters should not be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the disclosure are approximations, the numerical values set forth in the specific embodiments are reported as precisely as possible. The numerical values presented in some embodiments of the present disclosure may include certain errors necessarily caused by standard deviations found in their respective test measurements.

The grouping of alternative elements or embodiments of the present disclosure disclosed herein should not be construed as limiting. Each group element may be cited and claimed alone or in any combination with other elements of the group or other elements found herein. For convenience and/or patentability reasons, one or more elements of a group may be included in or deleted from the group. When any such inclusion or deletion occurs, the specification is considered herein to include the modified group, thereby satisfying the written description of all markush groups used in the appended claims.

The present disclosure is explained by various examples, which are intended to be purely examples of the present disclosure, and should not be considered as limiting the disclosure in any way. Various examples are provided to better illustrate the claimed disclosure and should not be construed as limiting the scope of the disclosure. To the extent that specific materials are mentioned, they are for illustrative purposes only and are not intended to limit the present disclosure. Those skilled in the art may develop equivalent means or reactants without the practice of the inventive capabilities and without departing from the scope of the present disclosure.

Although the application has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that embodiments of the application extend beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and modifications and equivalents thereof.

Claims

1. An apparatus for cell analysis, comprising:

a flow cell, a light source, a focusing module, a condenser lens, and one, two or more detectors,

The flow chamber is configured to form a sample stream of a measurement sample, the measurement sample comprising particles and/or cells; the light source is configured to emit illumination light for illuminating the sample stream; the focusing module is configured to direct reflected light of the illumination light away from the light source or block reflected light of the illumination light from entering the light source; the condenser lens is configured to collect either or both scattered light or fluorescence from particles and/or cells in the sample stream; the one, two or more detectors are configured to detect a signal of the scattered light and/or a signal of the fluorescence.

2. The apparatus of claim 1, wherein,

The light source further includes a receiving module configured to split the scattered light and the fluorescence collected by the condenser lens into two separate light paths toward two separate detectors, respectively.

3. The apparatus of claim 1, wherein,

The focusing module includes a lens that is either non-coaxial or non-perpendicular to a central axis of the illumination light.

4. The apparatus of claim 1, wherein,

The flow cell includes a surface illuminated by the illumination light and positioned non-perpendicularly to a central axis of the illumination light.

5. The apparatus of claim 1, wherein,

The flow chamber is part of a cartridge device and the cartridge device is configured to mix a sample with a reagent to form the measurement sample and form a sample flow of the measurement sample within the flow chamber.

6. The apparatus of claim 1, wherein,

A beam blocker is also included and positioned in the optical path between the flow chamber and one of the detectors to block illumination light passing through the flow chamber.

7. The apparatus of claim 1, wherein,

Also included is an aperture made of an opaque material and having a transparent opening in the center, the aperture positioned in front of one of the detectors to block light outside the transparent opening from entering the detector.

8. The apparatus of claim 1, wherein,

The focusing module is configured to focus the illumination light to form an elliptical spot on the sample flow, a major axis of the elliptical spot being perpendicular to a direction of the sample flow and a minor axis of the elliptical spot being along the direction of the sample flow, the major axis of the elliptical spot being greater than a width of the flow cell.

9. The apparatus of claim 8, wherein,

The focusing module includes at least one cylindrical lens and the flow cell is positioned at or near the focal point of one of the cylindrical lenses.

10. A method for cellular analysis, comprising:

Using the flow chamber to form a sample stream of the measurement sample; using a light source to emit illumination light; illuminating the sample stream with the illumination light; directing reflected light of the illumination light away from the light source or blocking reflected light of the illumination light from entering the light source; using a condensing lens to collect scattered light, or fluorescence, or both scattered light and fluorescence from particles and/or cells in the sample stream; one, two or more detectors are used to detect the signal of the scattered light and/or the signal of the fluorescence.

11. The method of claim 10, wherein,

A receiving module is used to split the scattered light and the fluorescence collected by the condenser lens into two separate light paths towards two separate detectors, respectively.

12. The method of claim 11, wherein,

The two separate detectors are used to detect the signal of the scattered light and the signal of the fluorescence.

13. The method of claim 10, wherein,

Further comprising focusing the illumination light using a focusing module to form an elliptical spot on the sample flow, the elliptical spot having a major axis perpendicular to the direction of the sample flow and a minor axis along the direction of the sample flow, the elliptical spot having a major axis greater than the width of the flow cell.

14. The method of claim 10, wherein,

The flow chamber is part of a cartridge device, further comprising mixing a sample with a reagent using the cartridge device to form the measurement sample and forming a sample stream of the measurement sample within the flow chamber.

15. The method of claim 14, wherein,

The reagent comprises a fluorescent labeling compound comprising an antibody bound to a fluorescent group, an antibody bound to a fluorescent particle or a fluorescent dye, or a combination thereof.

16. The method of claim 10, wherein,

Further comprising blocking illumination light passing through the flow chamber using a beam blocker.

17. The method of claim 14, wherein,

Further comprising placing the cartridge device in an analyzer device comprising a light source, a condenser lens and a detector to measure particles and/or cells of a sample flow within the flow chamber.

18. The method of claim 17, wherein,

Further comprising removing the cartridge device from the analyzer device after the measurement is completed.